Recombinant hexose oxidase, a method of producing same and use of such enzyme

ABSTRACT

A method of producing hexose oxidase by recombinant DNA technology, recombinant hexose oxidase and the use of such enzyme, in particular in the manufacturing of food products such as doughs and dairy products, animal feed, pharmaceuticals, cosmetics, dental care products and in the manufacturing of lactones. Suitable sources of DNA coding for the enzyme are marine algal species including  Chondrus crispus, Iridophycus flaccidum  and  Euthora cristata . In useful embodiments, the recombinant hexose oxidase is produced by  Pichia pastoris, Saccharomyces cerevisiae  or  E. coli.

This application is a continuation of U.S. application Ser. No.09/824,053, filed Apr. 3, 2001, now U.S. Pat. No. 6,924,366, which is adivisional of U.S. application Ser. No. 08/669,304, filed Sep. 11, 1996,now U.S. Pat. No. 6,251,626, which is a national stage entry of PCTinternational application PCT/DK96/00238, which is acontinuation-in-part of U.S. application Ser. No. 08/476,910, filed Jun.7, 1995, each of which is incorporated by reference in its entirety.

FIELD OF INVENTION

The invention provides a method of producing hexose oxidase byrecombinant DNA technology and such enzyme produced by the method andits use in the food industry and other fields.

TECHNICAL BACKGROUND AND PRIOR ART

Hexose oxidase (D-hexose:O₂-oxidoreductase, EC 1.1.3.5) is an enzymewhich in the presence of oxygen is capable of oxidizing D-glucose andseveral other reducing sugars including maltose, lactose and cellobioseto their corresponding lactones with subsequent hydrolysis to therespective aldobionic acids. Accordingly, hexose oxidase differ fromanother oxidoreductase, glucose oxidase which can only convert D-glucosein that this enzyme can utilize a broader range of sugar substrates. Theoxidation catalyzed by hexose oxidase can e.g. be illustrated asfollows:D-Glucose+O₂→δ-D-gluconolactone+H₂O₂, orD-Galactose+O₂→γ-D-galactogalactone+H₂O₂

Up till now, hexose oxidase (in the following also referred to as HOX)has been provided by isolating the enzyme from several red algal speciessuch as Iridophycus flaccidum (Bean and Hassid, 1956) and Chondruscrispus (Sullivan et al. 1973). Additionally, the algal species Euthoracristata has been shown to produce hexose oxidase.

It has been reported that hexose oxidase isolated from these naturalsources may be of potential use in the manufacturing of certain foodproducts. Thus, hexose oxidase isolated from Iridophycus flaccidum hasbeen shown to be capable of converting lactose in milk with theproduction of the corresponding aldobionic acid and has been shown to beof potential interest as an acidifying agent in milk, e.g. to replaceacidifying microbial cultures for that purpose (Rand, 1972). In thatrespect, hexose oxidase has been mentioned as a more interesting enzymethan glucose oxidase, since this latter enzyme can only be utilized inmilk or food products not containing glucose with the concomitantaddition of glucose or, in the case of a milk product, thelactose-degrading enzyme lactase, whereby the lactose is degraded toglucose and galactose. Even if glucose in this manner will becomeavailable as a substrate for the glucose oxidase, it is obvious thatonly 50% of the end products of lactase can be utilized as substrate bythe glucose oxidase, and accordingly glucose oxidase is not an efficientacidifying agent in natural milk or dairy products.

The capability of oxygen oxidoreductases including that of hexoseoxidase to generate hydrogen peroxide, which has an antimicrobialeffect, has been utilized to improve the storage stability of certainfood products including cheese, butter and fruit juice as it isdisclosed in JP-B-73/016612. It has also been suggested thatoxidoreductases may be potentially useful as oxygen scavengers orantioxidants in food products.

Within the bakery and milling industries it is known to use oxidizingagents such as e.g. iodates, peroxides, ascorbic acid, K-bromate orazodicarbonamide for improving the baking performance of flour toachieve a dough with improved stretchability and thus having a desirablestrength and stability. The mechanism behind this effect of oxidizingagents is that the flour proteins, such as e.g. gluten in wheat flourcontains thiol groups which, when they become oxidized, form disulphidebonds whereby the protein forms a more stable matrix resulting in abetter dough quality and improvements of the volume and crumb structureof the baked products.

However, such use of several of the currently available oxidizing agentsare objected to by consumers or is not permitted by regulatory bodiesand accordingly, it has been attempted to find alternatives to theseconventional flour and dough additives and the prior art has suggestedthe use of glucose oxidase for the above purpose. Thus, U.S. Pat. No.2,783,150 discloses the addition of glucose oxidase to flour to improvethe Theological characteristics of dough. CA 2,012,723 discloses closesbread improving agents comprising cellulolytic enzymes and glucoseoxidase and JP-A-084848 suggests the use of a bread improvingcomposition comprising glucose oxidase and lipase.

However, the use of glucose oxidase as a dough and bread improvingadditive has the limitation that this enzyme requires the presence ofglucose as substrate in order to be effective in a dough system andgenerally, the glucose content in cereal flours is low. Thus, in wheatflour glucose is present in an amount which is in the range of 0-0.4%w/w, i.e. flours may not contain any glucose at all. Therefore, theabsence or low content of glucose in doughs will be a limiting factorfor the use of glucose oxidase as a dough improving agent. In contrast,the content of maltose is significantly higher already in the freshlyprepared dough and further maltose is formed in the dough due to theactivity of β-amylase either being inherently present in the flour orbeing added.

The current source of hexose oxidase is crude or partially purifiedenzyme preparations isolated by extraction from the above nativelyoccurring marine algal species. However, since the amount of hexoseoxidase in algae is low, it is evident that a production of the enzymein this manner is too tedious and costly to warrant a cost effectivecommercial production of the enzyme from these natural sources.Furthermore, the provision of a sufficiently pure enzyme product at acost effective level is not readily achievable in this manner.

A considerable industrial need therefore exists to provide analternative and more cost effective source of this industrially valuableenzyme without being dependent on a natural source and also to providethe enzyme in a pure form, i.e. without any contaminating enzymeactivities or any other undesirable contaminating substances includingundesirable algal pigments and environmental pollutants which may bepresent in the marine areas where the hexose oxidase-producing algalspecies grow.

Furthermore, the industrial availability of a food grade quality ofhexose oxidase in sufficient amounts and at cost effective prices willundoubtedly open up for new applications of that enzyme not only in thefood industry, but also in other industrial areas as it will bediscussed in the following. One example of such a novel application ofthe recombinant hexose oxidase in the food industry is the use hereof asa dough improving agent, another example being the use of hexose oxidaseactive polypeptide or a recombinant organism producing the polypeptidein the manufacturing of lactones.

SUMMARY OF THE INVENTION

The present invention has, by using recombinant DNA technology, for thefirst time made it possible to provide hexose oxidase activepolypeptides in industrially appropriate quantities and at a quality andpurity level which renders the hexose oxidase active polypeptideaccording to the invention highly suitable for any relevant industrialpurpose including the manufacturing of food products andpharmaceuticals.

Accordingly, the invention pertains in a first aspect to a method ofproducing a polypeptide having hexose oxidase activity, comprisingisolating or synthesizing a DNA fragment encoding the polypeptide,introducing said DNA fragment into an appropriate host organism in whichthe DNA fragment is combined with an appropriate expression signal forthe DNA fragment, cultivating the host organism under conditions leadingto expression of the hexose oxidase active polypeptide and recoveringthe polypeptide from the cultivation medium or from the host organism.

In a further aspect, the invention relates to a polypeptide in isolatedform having hexose oxidase activity, comprising at least one amino acidsequence selected from the group consisting of

(i) Tyr-Glu-Pro-Tyr-Gly-Gly-Val- (SEQ ID NO:1) Pro, (ii)Ala-Ile-Ile-Asn-Val-Thr-Gly- (SEQ ID NO:2) Leu-Val-Glu-Ser-Gly-Tyr-Asp-X-X-X-Gly-Tyr-X-Val-Ser-Ser, (iii) Asp-Leu-Pro-Met-Ser-Pro-Arg- (SEQ IDNO:3) Gly-Val-Ile-Ala-Ser-Asn-Leu- X-Phe, (iv)Asp-Ser-Glu-Gly-Asn-Asp-Gly- (SEQ ID NO:4) Glu-Leu-Phe-X-Ala-His-Thr,(v) Tyr-Tyr-Phe-Lys, (SEQ ID NO:5) (vi) Asp-Pro-Gly-Tyr-Ile-Val-Ile-(SEQ ID NO:6) Asp-Val-Asn-Ala-Gly-Thr-X- Asp, (vii)Leu-Gln-Tyr-Gln-Thr-Tyr-Trp- (SEQ ID NO:7) Gln-Glu-Glu-Asp, (viii)X-Ile-Arg-Asp-Phe-Tyr-Glu- (SEQ ID NO:8) Glu-Met,where X represents an amino acid selected from the group consisting ofAla, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx, Gly, His, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, and muteins and variantshereof.

In still further aspects the invention relates to a recombinant DNAmolecule comprising a DNA fragment coding for a polypeptide havinghexose oxidase activity and to a microbial cell comprising such arecombinant DNA molecule.

In other aspects, the invention pertains to the use of the above hexoseoxidase active polypeptide or a microbial cell expressing such apolypeptide in the manufacturing of a food product or an animal feed andthe manufacturing of a pharmaceutical product, a cosmetic or a toothcare product.

In other useful aspects there is provided a method of reducing the sugarcontent of a food product, comprising adding to the food product anamount of the polypeptide or the microbial cell as disclosed herein,which is sufficient to remove at least part of the sugar initiallypresent in said food product, a method of preparing a baked product froma dough, comprising adding the hexose oxidase active polypeptide or amicrobial cell expressing such a polypeptide to the dough, and a doughimproving composition comprising the polypeptide or the microbial cellaccording to the invention and at least one conventional doughcomponent.

In another aspect, the invention relates to the use of the polypeptideor a microbial cell according to the invention as an analytical reagentfor measuring the content of sugars.

In an interesting aspect, the invention also provides the use of apolypeptide or a microbial cell according to the invention in themanufacturing of a lactone, whereby the polypeptide and/or the microbialcell is applied to a reactor containing a carbohydrate which can beoxidized by the polypeptide and operating the reactor under conditionswhere the carbohydrate is oxidized.

DETAILED DISCLOSURE OF THE INVENTION

Hexose oxidases are produced naturally by several marine algal species.Such species are i.a. found in the family Gigartinaceae which belong tothe order Gigartinales. Examples of hexose oxidase producing algalspecies belonging to Gigartinaceae are Chondrus crispus and Iridophycusflaccidum. Also algal species of the order Cryptomeniales including thespecies Euthora cristata are potential sources of the hexose oxidaseactive polypeptide according to the invention. Accordingly, such algalspecies are potentially useful sources of hexose oxidase and of DNAcoding for such hexose oxidase active polypeptides. As used herein theterm “hexose oxidase active polypeptide” denotes an enzyme which atleast oxidizes D-glucose, D-galactose, D-mannose, maltose, lactose andcellobiose.

When using such natural sources for the isolation of native hexoseoxidase, as it has been done in the prior art and in the presentinvention with the purpose of identifying algal material which could beused as a source of mRNA for use in the construction of cDNA and as thestarting point for constructing primers of synthetic DNAoligonucleotides, the enzyme is typically isolated from the algalstarting material by extraction using an aqueous extraction medium.

As starting material for such an extraction the algae may be used intheir fresh state as harvested from the marine area of growth or theymay be used after drying the fronds e.g. by air-drying at ambienttemperatures or by any appropriate industrial drying method such asdrying in circulated heated air or by freeze-drying. In order tofacilitate the subsequent extraction step, the fresh or dried startingmaterial may be comminuted e.g. by grinding or blending.

As the aqueous extraction medium, buffer solutions having a pH in therange of 6-8, such as 0.1 M sodium phosphate buffer, 20 mMtriethanolamine buffer or 20 mM Tris-HCl buffer are suitable. The hexoseoxidase is typically extracted from the algal material by suspending thestarting material in the buffer and keeping the suspension at atemperature in the range of 0-20° C. such as at about 5° C. for 1 to 10days, preferably under agitation.

The suspended algal material is then separated from the aqueous mediumby an appropriate separation method such as filtration, sieving orcentrifugation and the hexose oxidase subsequently recovered from thefiltrate or supernatant. Optionally, the separated algal material issubjected to one or more further extraction steps.

Since several marine algae contain coloured pigments such asphycocyanins, it may be required to subject the filtrate or supernatantto a further purification step whereby these pigments are removed. As anexample, the pigments may be removed by treating the filtrate orsupernatant with an organic solvent in which the pigments are solubleand subsequently separating the solvent containing the dissolvedpigments from the aqueous medium.

The recovery of hexose oxidase from the aqueous extraction medium can becarried out by any suitable conventional methods allowing isolation ofproteins from aqueous media. Such methods, examples of which will bedescribed in details in the following, include such methods as ionexchange chromatography, optionally followed by a concentration stepsuch as ultrafiltration. It is also possible to recover the enzyme byadding substances such as e.g. (NH₄)₂SO₄ which causes the protein toprecipitate, followed by separating the precipitate and optionallysubjecting it to conditions allowing the protein to dissolve.

For the purpose of the invention it is desirable to provide the enzymein a substantially pure form e.g. as a preparation essentially withoutother proteins or non-protein contaminants and accordingly, therelatively crude enzyme preparation resulting from the above extractionand isolation steps is preferably subjected to further purificationsteps such as further chromatography steps, gel filtration orchromatofocusing as it will also be described by way of example in thefollowing.

As it is mentioned above, the hexose oxidase active polypeptideaccording to invention is provided by means of recombinant DNAtechnology methods allowing it to be produced by cultivating in aculturing medium an appropriate host organism cell comprising a genecoding for the hexose oxidase, and recovering the enzyme from the cellsand/or the culturing medium.

The method of producing hexose oxidase which is provided hereincomprises as a first step the isolation or the construction of a DNAfragment coding for hexose oxidase. Several strategies for providingsuch a DNA fragment are available. Thus, the DNA fragment can beisolated as such from an organism which inherently produces hexoseoxidase. In order to identify the location of the coding DNA fragment,it is required to dispose of RNA or DNA probe sequences which underappropriate conditions will hybridize to the DNA fragment searched forand subsequently isolating a DNA fragment comprising the coding sequenceand cloning it in a suitable cloning vector.

Another suitable strategy, which is disclosed in details in the belowexamples, is to isolate mRNA from an organism producing the hexoseoxidase and use such mRNA as the starting point for the construction ofa cDNA library which can then be used for polymerase chain reaction(PCR) synthesis of DNA based on oligonucleotide primers which aresynthesized based on amino acid sequences of the hexose oxidase. It wasfound that such a strategy is suitable for providing a hexoseoxidase-encoding DNA fragment. By way of example such a strategy asdescribed in details below is described summarily.

Synthetic oligonucleotides were prepared based on the HOX-2 and HOX-3peptide sequences prepared as described hereinbelow by endoLys-Cdigestion of a 40 kD polypeptide of hexose oxidase extracted fromChondrus crispus. PCR using first strand cDNA as template and with asense HOX-2 primer and an anti-sense HOX-3 primer produced a DNAfragment of 407 bp. This fragment was inserted into an E. coli vector,pT7 Blue and subsequently sequenced. It was found that in addition tothe sequences for the HOX-2 and HOX-3 peptides this 407 bp fragment alsocontained an open reading frame containing the HOX-4 and HOX-5 peptidesof the above 40 kD Chondrus crispus-derived hexose oxidase fragment theisolation of which is also described in the following.

Sense and anti-sense oligonucleotides were synthesized based on the 407bp fragment, and two fragments of 800 and 1400 bp, respectively couldsubsequently be isolated by PCR using cDNA as template. These twofragments were cloned in the pT7 Blue vector and subsequently sequenced.The DNA sequence of the 5′-fragment showed an open reading framecontaining the HOX-6 peptide which was also isolated from the above 40kD Chondrus crispus-derived hexose oxidase fragment. Similarly, the3′-fragment showed a reading frame containing the HOX-1, the isolationof which is disclosed below, and the HOX-7 and HOX-8, both isolated froma 29 kD Chondrus crispus-derived hexose oxidase polypeptide obtained byendoLys-C digestion as also described in the following.

Based upon the combined DNA sequences as mentioned above, anoligonucleotide corresponding to the 5′-end of the presumed hox gene andan oligonucleotide corresponding to the 3′-end of that gene weresynthesized. These two oligonucleotides were used in PCR using firststrand cDNA as template resulting in a DNA fragment of about 1.8 kb.This fragment was cloned in the above E. coli vector and sequenced. TheDNA sequence was identical to the combined sequence of the above 5′-end,407 bp and 3′-end sequences and it was concluded that this about 1.8 kbDNA sequence codes for both the 40 kD and the 29 kD Chondruscrispus-derived hexose oxidase fragments.

As will be evident for the skilled artisan, the above strategy forisolating a DNA fragment encoding a hexose oxidase active polypeptide,including the isolation and characterization of the hexose oxidase, canbe used for the construction of such fragments encoding hexose oxidasesderived from any other natural source than Chondrus crispus includingthe marine algal species mentioned above, such as from other plants orfrom a microorganism.

Alternatively, the DNA sequence of the hexose oxidase activepolypeptide-encoding DNA fragment may be constructed synthetically byestablished standard methods e.g. the phosphoamidite method described byBeaucage and Caruthers (1981), or the method described by Matthes et al.(1984). According to the phosphoamidite method, oligonucleotides aresynthesized, eg in an automatic DNA synthesizer, purified, annealed,ligated and cloned in an appropriate vector.

Furthermore, the DNA fragment may be of mixed genomic and synthetic,mixed synthetic and cDNA or mixed genomic and cDNA origin, prepared byligating sub-fragments of synthetic, genomic or cDNA origin asappropriate, the sub-fragments corresponding to various parts of theentire DNA fragment, in accordance with standard techniques.

In a subsequent step of the method according to the invention, theisolated or synthesized hexose oxidase active polypeptide-encoding DNAfragment is introduced into an appropriate host organism in which theDNA fragment is combined operably with an appropriate expression signalfor the DNA fragment. Such an introduction can be carried out by methodswhich are well-known to the skilled practitioner including theconstruction of a vector having the fragment inserted and transformingthe host organism with the vector. Suitable vectors include plasmidswhich are capable of replication in the selected host organism. It isalso contemplated that the DNA fragment can be integrated into thechromosome of the host organism e.g. by inserting the fragment into atransposable element such as a transposon, and subjecting a mixture ofthe selected host organism and the transposon to conditions where thetransposon will become integrated into the host organism chromosome andcombine with an appropriate expression signal.

According to the invention, a hexose oxidase active polypeptide-encodingDNA fragment including the gene for the polypeptide, which is producedby methods as described above, or any alternative methods known in theart, can be expressed in enzymatically active form using an expressionvector. An expression vector usually includes the components of atypical cloning vector, i.e. an element that permits autonomousreplication of the vector in the selected host organism and one or morephenotypic markers for selection purposes. An expression vector includescontrol sequences encoding a promoter, operator, ribosome binding site,translation initiation signal and optionally, a repressor gene or one ormore activator genes. To permit the secretion of the expressedpolypeptide, a signal sequence may be inserted upstream of the codingsequence of the gene. In the present context, the term “expressionsignal” includes any of the above control sequences, repressor oractivator sequences and signal sequence. For expression under thedirection of control sequences, the hexose oxidase encoding gene isoperably linked to the control sequences in proper manner with respectto expression. Promoter sequences that can be incorporated into plasmidvectors, and which can support the transcription of the hexose oxidasegene include, but are not limited to the tac promoter, phagelambda-derived promoters including the P_(L) and P_(R) promoters.

An expression vector carrying the DNA fragment of the invention may beany vector which is capable of expressing the hexose oxidase gene in theselected host organism, and the choice of vector will depend on the hostcell into which it is to be introduced. Thus, the vector may be anautonomously replicating vector, i.e. a vector which exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid, a bacteriophage or anextrachromosomal element, a minichromosome or an artificial chromosome.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome.

In the vector, the DNA fragment coding for the hexose oxidase activepolypeptide should be operably combined with a suitable promotersequence. The promoter may be any DNA sequence which conferstranscriptional activity to the host organism of choice and may bederived from genes encoding proteins which are either homologous orheterologous to the host organism. Examples of suitable promoters fordirecting the transcription of the DNA fragment of the invention in abacterial host are the promoter of the lac operon of E. coli, theStreptomyces coelicolor agarase gene dagA promoters, the promoters ofthe Bacillus licheniformis α-amylase gene (amyL), the promoters of theBacillus stearothermophilus maltogenic amylase gene (amyM), thepromoters of the Bacillus amyloliquefaciens α-amylase gene (amyQ), thepromoters of the Bacillus subtilis xylA and xylB genes.

For transcription in a fungal species, examples of useful promoters arethose derived from the genes encoding the Pichia pastoris alcoholoxidase, Aspergillus oryzae TAKA amylase, Rhizomucor miehei asparticproteinase, Aspergillus niger neutral α-amylase, A. niger acid stableα-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, Aspergillusoryzae alkaline protease, Aspergillus oryzae triose phosphate isomeraseor Aspergillus nidulans acetamidase. As examples of suitable promotersfor expression in a yeast species the Gal 1 and Gal 10 promoters ofSaccharomyces cerevisiae can be mentioned. When expressed in a bacterialspecies such as E. coli, a suitable promoter may be selected from abacteriophage promoter including a T7 promoter or a lambda bacteriophagepromoter.

The vector comprising the DNA fragment encoding the hexose oxidaseactive polypeptide may also comprise a selectable marker, e.g. a genethe product of which complements a defect in the host organism such as amutation conferring an auxothrophic phenotype, or the marker may be onewhich confers antibiotic resistance or resistance to heavy metal ions.

The host organism of the invention either comprising a DNA construct oran expression vector as described above is advantageously used as a hostcell in the recombinant production of a polypeptide according to theinvention. The cell may be transformed with a DNA construct comprisingthe gene coding for the polypeptide of the invention or, conveniently byintegrating the DNA construct into the host chromosome. Such anintegration is generally considered to be advantageous as the DNAfragment is more likely to be stably maintained in the cell. Integrationof the DNA constructs into the host chromosome may be carried outaccording to conventional methods such as e.g. by homologous orheterologous recombination or by means of a transposable element.Alternatively, the host organism may be transformed with an expressionvector as described above.

In accordance with the invention, the host organism may be a cell of ahigher organism such as an animal cell, including a mammal, an avian oran insect cell, or a plant cell. However, in preferred embodiments, thehost organism is a microbial cell, e.g. a bacterial or a fungal cellincluding a yeast cell.

Examples of suitable bacterial host organisms are gram positivebacterial species such as Bacillaceae including Bacillus subtilis,Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillusstearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens,Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillusmegaterium and Bacillus thuringiensis, Streptomyces species such asStreptomyces murinus, lactic acid bacterial species includingLactococcus spp. such as Lactococcus lactis, Lactobacillus spp.including Lactobacillus reuteri, Leuconostoc spp. and Streptococcus spp.Alternatively, strains of a gram negative bacterial species such as aspecies belonging to Enterobacteriaceae, including E. coli or toPseudomonadaceae may be selected as the host organism.

A yeast host organism may advantageously be selected from a species ofSaccharomyces including Saccharomyces cerevisiae or a species belongingto Schizosaccharomyces. Suitable host organisms among filamentous fungiinclude species of Aspergillus, eg Aspergillus oryzae, Aspergillusnidulans or Aspergillus niger. Alternatively, strains of a Fusariumspecies, eg Fusarium oxysporum or of a Rhizomucor species such asRhizomucor miehei can be used as the host organism. In one preferredembodiment a strain of the species Pichia pastoris is used as hostorganism.

Some of the above useful host organisms such as fungal species or grampositive bacterial species may be transformed by a process which includeprotoplast formation and transformation of the protoplasts followed byregeneration of the cell wall in a manner known per se.

For the production of the hexose oxidase active polypeptide, therecombinant host organism cells as described above are cultivated underconditions which lead to expression of the polypeptide in a recoverableform. The medium used to cultivate the cells may be any conventionalmedium suitable for growing the host cells in question and obtainingexpression of the polypeptide. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes.

The resulting polypeptide is typically recovered from the cultivationmedium by conventional procedures including separating the cells fromthe medium by centrifugation or filtration, if necessary, afterdisruption of the cells, followed by precipitating the proteinaceouscomponents of the supernatant or filtrate e.g. by adding a salt such asammonium sulphate, followed by a purification step.

It is an industrially convenient aspect of the invention that microbialcultures such as e.g. bacterial cultures which are used in themanufacturing of food or feed products can be used as the host organismexpressing the gene coding for the hexose oxidase active polypeptide.Thus, lactic acid bacterial starter cultures which are used in themanufacturing of dairy products or other food products such as meatproduct or wine and which e.g. comprise one or more strains of a lacticacid bacterium selected from any of the above lactic acid bacterialspecies can be used as host organisms whereby the hexose oxidase will beproduced directly in the food product to which the starter cultures areadded.

Similarly, the hexose oxidase encoding gene according to the inventionmay be introduced into lactic acid bacterial starter cultures which areused as inoculants added to fodder crops such as grass or corn or toproteinaceous waste products of animal origin such as fish andslaughterhouse waste materials for the production of silage for feedingof animals. For this purpose, the expression of hexose oxidase by thesilage inoculants will imply that the oxygen initially present in thecrops or the waste materials to be ensiled is depleted whereby anaerobicconditions, which will inhibit growth of aerobic spoilage organisms suchas gram negative bacteria and yeasts, will be established.

It is also contemplated that yeast cultures such as baker's yeast oryeast cultures which are used in the manufacturing of alcoholicbeverages including wine and beer can be used as host organisms for thegene coding for the hexose oxidase active polypeptide of the invention.For example in the case of such recombinant baker's yeast strains, thehexose oxidase being produced will have a dough improving effect as itis described in the following.

From the above it is apparent that the direct addition of recombinantmicrobial cultures expressing the hexose oxidase according to theinvention to a food product or any other product where hexose oxidaseactivity is desired, can be used as an alternative to the addition ofthe isolated enzyme.

In further industrially important embodiments, the recombinant microbialcultures expressing a hexose oxidase active polypeptide are used in abioreactor for the production of the enzyme or for the production oflactones from either of the above-mentioned carbohydrates which can beoxidized by the hexose oxidase active enzyme. For this latterapplication, the cells of the microbial cultures are advantageouslyimmobilized on a solid support such as a polymer material, which ispreferable in the form of small particles to provide a large surface forbinding the cells. Alternatively, the isolated enzyme may be used forthe above purpose, also preferable bound to a solid support material. Inthis connection, the binding of the cells or the enzyme may be providedby any conventional method for that purpose.

In other useful embodiments of the invention, the polypeptide havinghexose oxidase activity may be a fusion product, i.e. a polypeptidewhich in addition to the hexose oxidase active amino acid sequencescomprises further amino acid sequences having other useful activities.Thus, fusion polypeptides having one or more enzyme activities inaddition to the hexose oxidase activity are contemplated. Suchadditional enzyme activities may be chosen among enzymes capable ofdegrading carbohydrates, such as lactase, amylases includingglucoamylases, glucanases, cellulases, hemicellulases, xylanases,lactases or any other oxidoreductase such as glucose oxidase, galactoseoxidase or pyranose oxidase, and also among proteases and peptidases,lipases or nucleases. The additional enzyme sequence(s) to be chosen forintegration into a hexose oxidase polypeptide according to the inventiondepend(s) on the product for which the enzymatically active fusionproduct is intended. Thus, as examples, it is contemplated that a hexoseoxidase active fusion polypeptide for use in the manufacturing of adairy product advantageously comprises a lactase, a protease or apeptidase, and that a fusion polypeptide intended for dough improvementmay as the fusion partner comprise any of the above carbohydratedegrading enzymes. It is also apparent that microbial cells according tothe invention as described above and which express a hexose oxidaseactive fusion polypeptide having additional enzyme activities may beused for inoculation of other food products and animal feeds in themanner as also described above.

It is also contemplated that a suitable fusion partner may be a sequenceconferring to the hexose oxidase altered characteristics such assolubility or a sequence which can serve as a “tagging” group conferringto the hexose oxidase the ability to bind more strongly or moreselectively to a particular solid material for hexose oxidasepolypeptide purification or immobilization purposes.

Furthermore, it is within the scope of the invention to provide thepolypeptide as a chimeric product comprising partial sequences of hexoseoxidase active polypeptides derived from different sources and beingencoded by a DNA fragment which is constructed by combining hexoseoxidase active polypeptide-encoding DNA sequences from these differentsources into one DNA fragment encoding the entire chimeric polypeptide.

In one useful embodiment the method according to the invention is onewherein the DNA fragment encoding the hexose oxidase active polypeptidecomprises at least one DNA sequence coding for an amino acid sequenceselected from the group consisting of

(i) Tyr-Glu-Pro-Tyr-Gly-Gly-Val- (SEQ ID NO:1) Pro, (ii)Ala-Ile-Ile-Asn-Val-Thr-Gly- (SEQ ID NO:2) Leu-Val-Glu-Ser-Gly-Tyr-Asp-X-X-X-Gly-Tyr-X-Val-Ser-Ser, (iii) Asp-Leu-Pro-Met-Ser-Pro-Arg- (SEQ IDNO:3) Gly-Val-Ile-Ala-Ser-Asn-Leu- X-Phe, (iv)Asp-Ser-Glu-Gly-Asn-Asp-Gly- (SEQ ID NO:4) Glu-Leu-Phe-X-Ala-His-Thr,(v) Tyr-Tyr-Phe-Lys, (SEQ ID NO:5) (vi) Asp-Pro-Gly-Tyr-Ile-Val-Ile-(SEQ ID NO:6) Asp-Val-Asn-Ala-Gly-Thr-X- Asp, (vii)Leu-Gln-Tyr-Gln-Thr-Tyr-Trp- (SEQ ID NO:7) Gln-Glu-Glu-Asp, (viii)X-Ile-Arg-Asp-Phe-Tyr-Glu- (SEQ ID NO:8) Glu-Met,where X represents an amino acid selected from the group consisting ofAla, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx, Gly, His, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val,and muteins and variants hereof.

In the present context, the term “variant” is used to designate anymodification of a hexose oxidase active polypeptide sequence which doesnot result in complete loss of the hexose oxidase activity. Themodifications may include deletion, substitution of amino acid residuespresent in the polypeptide as it is derived from a natural source or inan already modified polypeptide sequence or the modification may implythe insertion into such a polypeptide of additional amino acid residues.Substitution of one or more amino acid residues may be carried out bymodifying or substituting the codon or codons coding for the amino acidor amino acids which it is desired to substitute, e.g. by mutagenesis,in particular site-directed mutagenesis, using methods which are knownper se. Similarly, deletion of one or more amino acid residues can bemade by deleting the corresponding codon or codons in the DNA fragmentcoding for the polypeptide according to the invention.

As also mentioned above, the method according to the invention may as afurther step include a purification of the polypeptide preparationinitially recovered from the cultivation medium and/or themicroorganisms. The purpose of this further step is to obtain an enzymepreparation in which the hexose oxidase polypeptide is in asubstantially pure form. The term “substantially pure form” implies thatthe preparation is without any undesired contaminating substancesoriginating from the cultivation medium, the production host organismcells or substances produced by these cells during cultivation. Thus, itis for many applications of importance that the polypeptide preparationresulting from the purification step is substantially without anynon-hexose oxidase enzymatic activity. The purification methods willdepend on the degree of purity which is desirable, but will typically beselected from conventional protein purification methods such as saltingout, affinity or ion exchange chromatography procedures includinghydrophobic interaction chromatography, and gel filtration methods, suchas the method described in the following examples.

As mentioned above, the invention relates in a further aspect to apolypeptide in isolated form having hexose oxidase activity, comprisingat least one of the above amino acid sequences, or muteins and variantshereof as they are described above. Preferably, the polypeptide isproduced according to the methods as described above.

Depending on the method of production, in particular the host organismin question, the polypeptide according to the invention may beglycosylated to a varying degree or it may for certain purposesadvantageously be expressed in a substantially non-glycosylated form.

In one preferred embodiment of the invention, the polypeptide is onewhich has functional characteristics identical or partially identical tothose of hexose oxidase naturally occurring in the algal speciesChondrus crispus as they are described in the prior art. It was foundthat such a hexose oxidase extracted from the algal source when it wassubjected to SDS-PAGE as described herein may show separate proteinbands of 29, 40 and/or 60 kD.

In order to obtain a generally cost effective use of the polypeptide, itis preferred that the enzyme has a high enzymatic activity over a broadpH range. Thus, it is preferred that the hexose oxidase according to theinvention at least shows an enzymatic activity at a pH in the range of1-9, such as in the range of 2-9 including the range of 5-9. In thisconnection it is contemplated that the pH range of activity or the pHoptimum of a naturally derived hexose oxidase may be modified in adesired direction and to a desired degree by modifying the enzyme asdescribed above or by random mutagenesis of a replicon or a hostorganism comprising the DNA coding for the hexose oxidase, followed byselection of mutants having the desired altered pH characteristics.Alternatively, such modifications of the enzyme may be aimed atmodifying the thermotolerance and optimum temperature for activity ofthe hexose oxidase active polypeptide, or at changing the isoelectricpoint of the enzyme.

Furthermore, the polypeptide according to the invention is preferablyenzymatically active within a broad temperature range such as a range of10-90° C., e.g. within a range of 15-80° C. including the range of20-60° C. In particular, it may for certain specific purposes bepreferred that the hexose oxidase active polypeptide maintains asignificant residual enzymatic activity at temperatures of 70° C. orhigher, e.g. when the enzyme is intended for use in doughs where it maybe useful to have hexose oxidase activity during at least part of thesubsequent baking step.

The scope of application of the hexose oxidase depends on the range ofcarbohydrates which they can use as substrate. Although the hexoseoxidase appear to have highest substrate specificity for hexoses, suchas glucose, galactose and mannose, it has been found that the range ofcarbohydrate substances which can be utilized as substrates for thepolypeptide according to the invention is not limited to hexoses. Thus,a preferred polypeptide is one which in addition to a high specificityfor hexoses also has a high specificity for other carbohydratesubstances including disaccharides such as lactose, maltose and/orcellobiose and even also substantial specificity to pentoses includingas an example xylose, or deoxypentoses or deoxyhexoses such as rhamnoseor fucose. It is of significant practical implication that the hexoseoxidase in addition to a high specificity to hexoses and othermonosaccharides also has substantial specificity for disaccharides, inparticular lactose present in milk and maltose which i.a. occurs incereal flours and doughs.

Accordingly, in another preferred embodiment the polypeptide accordingto the invention is one which in addition to D-glucose oxidizes at leastone sugar selected from the group consisting of D-galactose, maltose,cellobiose, lactose, D-mannose, D-fucose and D-xylose.

In still another preferred embodiment the hexose oxidase activepolypeptide has an isoelectric point in the range of 4-5. Specifically,the polypeptide may preferably have an isoelectric point of 4.3±0.1 orone of 4.5±0.1.

Generally, the polypeptide according to the invention typically has amolecular weight as determined by gel filtration using Sephacryl S-200Superfine (Pharmacia) which is in the range of 100-150 kD, A molecularweight determined by this or equivalent methods are also referred to asan apparent molecular weight. Specifically, the polypeptide may have anapparent molecular weight of 110 kD±10 kD.

In a still further aspect, the invention provides a recombinant DNAmolecule comprising a DNA fragment coding for a polypeptide havinghexose oxidase activity. As it has been described above, such a DNAfragment may be isolated from a natural source or it may be constructede.g. as it is described in details in the below examples. Furthermore,the coding fragment may also be synthesized based upon amino acidsequences of a naturally occurring hexose oxidase. The recombinantmolecule can be selected from any of the expression vector types asmentioned above. In preferred embodiments, the recombinant DNA moleculecomprises a DNA fragment coding for a hexose oxidase polypeptide whichcomprises at least one of the above amino acid sequences (i) to (viii),or a mutein or derivative of such polypeptide. In one specificembodiment, the recombinant DNA molecule comprises the following DNAsequence (SEQ ID NO:30):

TGAATTCGTG GGTCGAAGAG CCCTTTGCCT CGTCTCTCTG 60 GTACCGTGTA TGTCAAAGGTTCGCTTGCAC ACTGAACTTC ACGATGGCTA CTCTTCCTCA 120 GAAAGACCCC GGTTATATTGTAATTGATGT CAACGCGGGC ACCGCGGACA AGCCGGACCC 180 ACGTCTCCCC TCCATGAAGCAGGGCTTCAA CCGCCGCTGG ATTGGAACTA ATATCGATTT 240 CGTTTATGTC GTGTACACTCCTCAAGGTGC TTGTACTGCA CTTGACCGTG CTATGGAAAA 300 GTGTTCTCCC GGTACAGTCAGGATCGTCTC TGGCGGCCAT TGCTACGAGG ACTTCGTATT 360 TGACGAATGC GTCAAGGCCATCATCAACGT CACTGGTCTC GTTGAGAGTG GTTATGACGA 420 CGATAGGGGT TACTTCGTCAGCAGTGGAGA TACAAATTGG GGCTCCTTCA AGACCTTGTT 480 CAGAGACCAC GGAAGAGTTCTTCCCGGGGG TTCCTGCTAC TCCGTCGGCC TCGGTGGCCA 540 CATTGTCGGC GGAGGTGACGGCATTTTGGC CCGCTTGCAT GGCCTCCCCG TCGATTGGCT 600 CAGCGGCGTG GAGGTCGTCGTTAAGCCAGT CCTCACCGAA GACTCGGTAC TCAAGTATGT 660 GCACAAAGAT TCCGAAGGCAACGACGGGGA GCTCTTTTGG GCACACACAG GTGGCGGTGG 720 CGGAAACTTT GGAATCATCACCAAATACTA CTTCAAGGAT TTGCCCATGT CTCCACGGGG 780 CGTCATCGCA TCAAATTTACACTTCAGCTG GGACGGTTTC ACGAGAGATG CCTTGCAGGA 840 TTTGTTGACA AAGTACTTCAAACTTGCCAG ATGTGATTGG AAGAATACGG TTGGCAAGTT 900 TCAAATCTTC CATCAGGCAGCGGAAGAGTT TGTCATGTAC TTGTATACAT CCTACTCGAA 960 CGACGCCGAG CGCGAAGTTGCCCAAGACCG TCACTATCAT TTGGAGGCTG ACATAGAACA 1020 GATCTACAAA ACATGCGAGCCCACCAAAGC GCTTGGCGGG CATGCTGGGT GGGCGCCGTT 1080 CCCCGTGCGG CCGCGCAAGAGGCACACATC CAAGACGTCG TATATGCATG ACGAGACGAT 1140 GGACTACCCC TTCTACGCGCTCACTGAGAC GATCAACGGC TCCGGGCCGA ATCAGCGCGG 1200 CAAGTACAAG TCTGCGTACATGATCAAGGA TTTCCCGGAT TTCCAGATCG ACGTGATCTG 1260 GAAATACCTT ACGGAGGTCCCGGACGGCTT GACTAGTGCC GAAATGAAGG ATGCCTTACT 1320 CCAGGTGGAC ATGTTTGGTGGTGAGATTCA CAAGGTGGTC TGGGATGCGA CGGCAGTCGC 1380 GCAGCGCGAG TACATCATCAAACTGCAGTA CCAGACATAC TGGCAGGAAG AAGACAAGGA 1440 TGCAGTGAAC CTCAAGTGGATTAGAGACTT TTACGAGGAG ATGTATGAGC CGTATGGCGG 1500 GGTTCCAGAC CCCAACACGCAGGTGGAGAG TGGTAAAGGT GTGTTTGAGG GATGCTACTT 1560 CAACTACCCG GATGTGGACTTGAACAACTG GAAGAACGGC AAGTATGGTG CCCTCGAACT 1620 TTACTTTTTG GGTAACCTGAACCGCCTCAT CAAGGCCAAA TGGTTGTGGG ATCCCAACGA 1680 GATCTTCACA AACAAACAGAGCATCCCTAC TAAACCTCTT AAGGAGCCCA AGCAGACGAA 1740 ATAGTAGGTC ACAATTAGTCATCGACTGAA GTGCAGCACT TGTCGGATAC GGCGTGATGG 1800 TTGCTTTTTA TAAACTTGGT A1801

Furthermore, the invention provides in another aspect a microbial cellwhich comprises the above recombinant DNA molecule. The above generaldescription of the host organism comprising a DNA fragment encoding thepolypeptide according to the invention encompasses such a microbial celland accordingly, such cells can be selected from any of the abovementioned microbial groups, families, genera and species, i.e. themicrobial cell may be selected from a bacterial cell, a fungal cell anda yeast cell including as examples an E. coli cell, a lactic acidbacterial cell, a Saccharomyces cerevisiae cell and a Pichia pastoriscell.

The microbial cell according to the invention may, if it is intended fordirect addition to a product where it is desired to have hexose oxidaseactivity, e.g. during a manufacturing process, be provided in the formof a microbial culture, preferable in a concentrate form. Thus, such aculture may advantageously contain the microbial cell according to theinvention in a concentration which is preferably in the range of 10⁵ to10¹² per g of culture. The culture may be a fresh culture, i.e. anon-frozen suspension of the cells in a liquid medium or it may in theform of a frozen or dried culture, e.g. a freeze-dried culture. Themicrobial cell may also for specific purposes be immobilized on a solidsubstrate.

As mentioned above, the invention relates in another further aspect tothe use of the hexose oxidase active polypeptide according to theinvention or of a microbial cell expressing such a polypeptide in themanufacturing of food products. In this context the term “manufacturing”should be understood in its broadest sense so as to encompass additionof the hexose oxidase or the microbial cell to ingredients for the foodproduct in question, prior to, during or after any subsequent processstep, during packaging and during storage of the finished product uptill it is consumed. The food products where such use is advantageousmay be any product where the end products of the hexose oxidase conferadvantageous effects on the food product.

Naturally, the desired activity of the hexose oxidase will only beobtained if substrate for the enzyme is present in sufficient amounts.The substrate carbohydrates may be inherently present in the foodproduct or the ingredients herefor or they may be added or generatedduring the manufacturing process. An example of substrate beinggenerated during manufacturing is the enzymatic degradation of di-,oligo- or polysaccharides to lower sugar substances which is degradableby the hexose oxidase which may occur as the result of enzymaticactivity of enzymes inherently present in the food product or addedduring the manufacturing. Furthermore, substrate for the hexose oxidaseactive polypeptide may be generated as the result of the enzymaticactivity of a fusion partner as described above.

The desirable effects of hexose oxidase activity in a product containingsubstrates for the enzyme include generation of lactones from the sugarsubstrate which may subsequently be converted to corresponding acids,generation of hydrogen peroxide and consumption of oxygen.

Typical examples of food products where hexose oxidase activity may beadvantageous include as examples dairy products, starch-containing foodproducts and non-dairy beverages. Thus, in the manufacturing of a rangeof dairy products it is desired to lower the pH. This is conventionallyobtained by inoculating the milk with lactic acid-producing startercultures. As mentioned above, it is contemplated that hexose oxidase ororganisms expressing this enzyme may be used as an alternative means ofacidifying milk. The same effect may be desirable in other food productswhich are acidified during manufacturing such as certain meat productsor vegetable product which are currently acidified by the addition oflactic acid bacterial starter cultures.

The consumption of oxygen resulting from the activity of the hexoseoxidase has several advantageous implications in relation to themanufacturing of food products and pharmaceuticals. By causing depletionor removal of oxygen in foods or pharmaceuticals containing lipids whichare prone to oxidative spoilage processes, the hexose oxidase may act asan antioxidant and additionally, the reduction of oxygen content mayinhibit spoilage organisms the growth of which is dependent on presenceof oxygen and accordingly, the hexose oxidase active polypeptide mayalso act as an antimicrobial agent.

This latter effect can be utilized to extend the shelf life of packagedfoods where spoilage can be prevented by the incorporation of the hexoseoxidase active polypeptide according to the invention either in the foodproduct itself or by providing a mixture of the hexose oxidase and anappropriate substrate herefor in the packaging, but separate from thecontent of food product. In a typical example, such a mixture isattached to the inner side of a food container such as eg a tin or ajar. Accordingly, the hexose oxidase according to the invention can beused as an oxygen removing agent in a food packaging.

It is evident that the above effects of the polypeptide according to theinvention in the manufacturing of food products will also be applicablein the manufacturing of animal feed products. In particular, theseeffects are desirable in the making of silage either made from foddercrops such as grass or corn or from proteinaceous animal waste productsfrom slaughterhouses or fish processing plants. Such feed products arecurrently ensiled by the addition of acids or acid producing bacteriasuch as lactic acid bacterial inoculants. In order to promote growth ofacidifying bacteria and to prevent the growth of aerobic spoilageorganisms such as gram negative bacteria and yeasts it is essential tohave a low oxygen content in the silage material. It is thereforecontemplated that the hexose oxidase according to the invention isuseful as oxygen removing and acidifying agent in the ensiling of feeds,optionally in the form of compositions further comprising one or moreconventional silage additive such as lactic acid bacterial inoculants orenzymes which generate low molecular sugar substances.

A further useful application of the hexose oxidase polypeptide accordingto the invention is the use of the enzyme to reduce the sugar content ofa food product, comprising adding to the product an amount of thepolypeptide or a microbial cell producing the polypeptide which issufficient to remove at least part of the sugar initially present in thefood product. Such an application may e.g. be useful in themanufacturing of diets for diabetic patients where a low sugar contentis desired, and in the production of wines with a reduced alcoholcontent. In this latter application, the hexose oxidase is preferablyadded to the must prior to yeast inoculation.

In a further useful aspect, the invention relates to the use of thehexose oxidase active polypeptide or of a microbial cell producing theenzyme according to the invention in the manufacturing of pharmaceuticalproducts, cosmetics or tooth care products such as tooth pastes ordentrifices. The desired effects of the hexose oxidase in such productsare essentially those described above with respect to food products andanimal feeds.

One particularly interesting use of the hexose oxidase according to theinvention is its use as a dough improving agent. It has been found thatthe addition of the hexose oxidase to a dough results in an increasedresistance hereof to breaking when the dough is stretched, i.e. theenzyme confers to the dough an increased strength whereby it becomesless prone to mechanical deformation. It is, based on the known effectsin this regard for glucose oxidase, contemplated that this effect ofaddition of the hexose oxidase according to the invention to a dough isthe result of the formation of cross-links between thiol groups insulphur-containing amino acids in flour proteins which occurs when thehydrogen peroxide generated by the enzyme in the dough reacts with thethiol groups which are hereby oxidized.

Accordingly, the invention also provides a method of preparing a bakedproduct from a dough, comprising adding to the dough an effective amountof the polypeptide or a microorganism according to the invention whichis capable of expressing such a polypeptide, and a dough improvingcomposition comprising the polypeptide or a microorganism capable ofexpressing such a polypeptide in a dough, and at least one conventionaldough component. In useful embodiments such a composition may furthercomprise at least one dough or bakery product improving enzyme e.g.selected from a cellulase, a hemicellulase, a pentosanase, a lipase, axylanase, an amylase, a glucose oxidase and a protease.

In still further aspects of the invention, the hexose oxidase is used asan analytical reagent in methods of determining in a biological andother samples the concentration of any sugar which can be converted bythe enzyme. Typically, the sugar content is measured by determining theamount of end products resulting from the enzymatic conversion of thesubstrate sugar present in the sample to be measured. In thisconnection, it is contemplated that the hexose oxidase can be useddirectly as a reagent in an in vitro analytical assay or that it can beincorporated in a sensor.

The invention will now be described by way of illustration in thefollowing examples and the annexed drawings of which:

FIG. 1 represents a schematic overview of the purification of hexoseoxidase (HOX) and the two strategies adopted for obtaining amino acidsequence information,

FIG. 2 shows native, non-dissociating polyacrylamide gel electrophoresis(native PAGE) of preparations of hexose oxidase at different steps ofthe purification. The samples represent the enzyme preparation obtainedafter anion exchange chromatography and concentration (lane 1), aftergel filtration (lane 2), and after either cation exchange chromatography(lane 3) or chromatofocusing (lane 4). The Phast gel (Pharmacia, 8-25%gradient gel) was silver stained. Molecular weights of standard proteins(×10⁻³) are indicated at the left. The band corresponding to hexoseoxidase, which is indicated by an arrow, was identified by enzymestaining of another gel in parallel (not shown). The four lanes were runon separate gels,

FIG. 3 shows the UV-profile obtained during purification of hexoseoxidase by gel filtration on Sephacryl S-200 HR as described in thetext. Fractions containing hexose oxidase (HOX) activity are indicatedby the filled area,

FIG. 4 shows SDS-PAGE of hexose oxidase purified from Chondrus crispusby anion exchange chromatography on DEAE-Sepharose Fast Flow, gelfiltration on Sephacryl S-200, followed by either cation exchangechromatography on S-Sepharose Fast Flow (lane 1) or chromatofocusing ona Mono P column (lane 2). Molecular weights of standard proteins (×10⁻³)are indicated at the left. The polypeptides at 60 kD, 40 kD and 29 kDare marked by arrows. Reduced samples were run on a 12% polyacrylamidegel which was stained with Coomassie massie Brilliant Blue R-250. Thetwo lanes were run on separate gels,

FIG. 5 shows isoelectric focusing (IEF) of hexose oxidase. The gel waseither stained with Coomassie Brilliant Blue R-250 (lane 1) or stainedfor enzyme activity as described in the text (lane 2). The positions ofisoelectric point markers run in parallel are shown at the left. The twolanes were run on separate gels,

FIG. 6 shows reversed phase HPLC separation of peptides generated bydigestion of the 40 kD HOX-polypeptide with endoproteinase Lys-C. Thepeaks labelled 1, 2, 3, 4 and 5 were subjected to amino acid sequencing,

FIG. 7 shows reversed phase HPLC separation of peptides generated bydigestion of the 29 kD HOX-polypeptide with endoproteinase Lys-C. Thepeaks labelled 1 and 2 were subjected to amino acid sequencing,

FIG. 8 shows a Northern blot analysis of RNA extracted from Chondruscrispus. The denaturing agarose gel was loaded with 30 μg (lane 1) and 3μg (lane 2), respectively of total RNA. Left arrow indicates hexoseoxidase specific transcript. The positions of molecular weight markersin kb are shown to the right,

FIG. 9 shows the construction of plasmid pUPO153 which mediates theexpression of recombinant hexose oxidase in Pichia pastoris. Smallarrows indicate PCR primers. The grey box indicates the hexose oxidasegene,

FIG. 10 shows purification of recombinant hexose oxidase from Pichiapastoris by anion exchange chromatography on HiTrap-Q column (step one).Alcohol oxidase (AOX) activity (●) and hexose oxidase (HOX) activity (∘)in the collected fractions were assayed as described in the text,

FIG. 11 shows purification of recombinant hexose oxidase from Pichiapastoris by gel filtration on Sephacryl S-200 HR (step two). Alcoholoxidase (AOX) activity (●) and hexose oxidase (HOX) activity (∘) in thecollected fractions were assayed as described in the text,

FIG. 12 shows the construction of plasmid pUPO181 which mediates theexpression of recombinant hexose oxidase in E. coli. Small arrowsindicate PCR primers (grey box indicates the hexose oxidase gene),

FIG. 13 shows SDS-PAGE of recombinant hexose oxidase produced in E.coli. Crude extracts from lysed cells were analyzed in a 14% denaturinggel. Molecular weights of standard proteins (×10⁻³) are indicated to theleft. The gel was stained with Coomassie Brilliant Blue R-250. Lane 1shows extract from E. coli cells with pUPO181, lane 2 shows plasmid-lesscontrol. Arrow shows hexose oxidase band and

FIG. 14 shows the construction of plasmid pUPO155 which mediates theexpression of recombinant hexose oxidase in Saccharomyces cerevisiae.Small arrows indicate PCR primers. The grey box indicates the hexoseoxidase gene.

EXAMPLE 1

Purification of Hexose Oxidase from Chondrus crispus

A schematic overview of the purification and two strategies adopted forobtaining amino acid sequence information for the enzyme is shown inFIG. 1.

1.1. Collection, Drying and Grinding of Chondrus crispus

The red sea-weed Chondrus crispus was collected during April toSeptember at the shore near Gren{dot over (a)}, Jutland, Denmark at adepth of 2-5 meters. Freshly collected algal fronds were rinsed withcold water and stored on ice during transport to the laboratory (<24hours). The sea-weed was then either dried immediately or stored infrozen state until further processing. For enzyme purification thematerial was stored at −18° C., whereas the material intended forisolation of mRNA was stored in liquid nitrogen.

Fronds of Chondrus crispus were thawed at 4° C. and air-dried at roomtemperature (20-25° C.) for 2-3 days. The dried material was ground tofine powder in a Waring Commercial Blendor (model 34BL97, Waring, NewHarford, Conn., USA).

1.2. Extraction of Enzyme

About 500 g of Chondrus crispus powder was mixed with 2.5 1 of 20 mMTris-Cl, pH 7.0. The water used throughout all extraction andpurification procedures was obtained from a Milli-Q UF Plus LaboratoryWater Purification System (Millipore). The buffer was pre-cooled to 4°C. The mixture was kept at 4° C. for 6-8 days. The extract was collectedby filtration through several layers of gauze.

The sea-weed material was subjected to repeated extractions which werecarried out as the first one described above. The material was usuallydiscarded after 5-8 extractions when residual activity had declined toan almost negligible level.

The filtrate was clarified by centrifugation at 10,000 ×g in a SorvallGSA rotor (Sorvall Instruments). The supernatant was filtered throughWhatman chromatography paper (chr 1) and diluted with water to aconductivity of 7-8 mS/cm. pH was adjusted to 7.5. The extract was thenready for anion exchange chromatography as described below.

1.3. Assay of Hexose Oxidase

The procedure used was essentially as described by Sullivan and Ikawa,1973. This assay is based on the principle that the hydrogen peroxideformed in the oxidation of the sugar in the presence of peroxidasereacts with the chromogenic substance, o-dianisidine to form a dye withabsorbance at 402 nm.

The assay mixture consisted of 1-40 μl of enzyme sample and 850 μl of anassay solution containing 370 μl of 0.1 M sodium phosphate buffer, pH7.0; 462 μl of 0.1 M D-glucose in 0.1 M sodium phosphate buffer, pH 7.0;9 μl of horse radish peroxidase, 0.1 mg/ml in water (Sigma Chemicals,cat. no. P 6782 or Boehringer Mannheim, cat. no. 814 393); and 9 μl ofo-dianisidine.2HCl, 3.0 mg/ml in water (3,3′-dimethoxybenz-idine, SigmaChemicals). After incubation at room temperature for 15 or 30 minutesthe assay was stopped by addition of one drop of 37% HCl (Merck, p.a.).Samples of 100 μl were transferred from the assay tubes to the wells ofa microtiter plate (NUNC, Denmark) and the absorbance at 410 nm was readon a Titertek Multiskan II PLUS plate reader (Labsystems/FlowLaboratories, Finland). To ensure that the observed activity was due tohexose oxidase—and not glucose oxidase—the assay was occasionallyperformed with D-galactose as the substrate instead of D-glucose.

1.4. Anion Exchange Chromatography

This step was carried out on a BioPilot chromatography system (PharmaciaBiotech, Sweden) connected to a SuperRac fraction collector(LKB-Produkter AB, Sweden).

This and the following steps in the purification were carried out atroom temperature (20-25° C.), but the fraction collector was placed in arefrigerator so that collected fractions were stored at 4° C. untilenzyme assay. Absorbance at 280 nm and conductivity were recorded. Theextract was applied onto a XK50/30 column (Pharmacia, 5.0×25 cm) with abed volume of 500 ml which had been packed with DEAE-Sepharose Fast Flow(Pharmacia) and equilibrated with buffer A: 20 mM Tris-Cl, pH 7.5. Theflow rate was 5 ml/min during sample application and 10 ml/min duringthe subsequent steps of the chromatography. After sample application,the column was washed with 1200 ml of buffer A. Adsorbed proteins wereeluted with 2800 ml of a gradient from 0% to 100% buffer B: 20 mMTris-Cl, 500 mM NaCl, pH 7.5. Fractions of 15 ml were collected duringthe gradient elution.

After each chromatographic run the column was regenerated with 500 ml of0.5 M NaOH, neutralised with 500 ml of 1.0 M Tris-Cl pH 7.5 and finallyequilibrated with 1200 ml of buffer A. The collected fractions wereassayed for hexose oxidase activity as described above (40 μl of sample,30 min of incubation time). Fractions of hexose oxidase activity werepooled and stored at 4° C.

1.5. Concentration of Hexose Oxidase Activity-Containing Fractions

Several pools of fractions from DEAE-Sepharose chromatography werepooled and concentrated by ultrafiltration in a Millipore LabUltrafiltration Cassette System (cat. no. XX420LCS0). The system wasequipped with a 30,000 nominal molecular weight limit (NMWL) membranecell (cat. no. PTTKOLCP2) and was driven by a peristaltic pump. Afterconcentration at room temperature to about 50 ml, the enzyme preparationwas further concentrated to 10-20 ml by centrifugal ultrafiltration at4° C. in Centriprep concentrators (Amicon, USA, nominal molecular weightcut-off 30,000) according to the instructions of the manufacturer. Theconcentrated enzyme solution was stored at 4° C.

1.6. Native Polyacrylamide Gel Electrophoresis (PAGE)

The composition of the preparation of hexose oxidase obtained by ionexchange chromatography and ultrafiltration was analyzed by native PAGEon a Pharmacia Phast System, see FIG. 2. The 8-25% gradient gels wererun and silver stained for protein according to the instructions of themanufacturer. A kit containing the following molecular weight markerswas also obtained from Pharmacia: Thyreglobulin (669,000); ferritin(440,000); catalase (232,000); lactate dehydrogenase (140,000) andalbumin (67,000).

Staining for hexose oxidase activity was carried out as described forglucose oxidase by Sock & Rohringer (1988). In principle, the redoxreaction catalyzed by glucose oxidase or hexose oxidase is coupled withreduction of tetrazolium salt to coloured, insoluble formazan.

Immediately after electrophoresis the Phast gel was submerged in 10 mlof freshly prepared staining solution containing: 0.1 M D-glucose (orD-galactose); 85 mM citric acid/sodium phosphate pH 6.5; 0.2 mg/ml of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(“thiazolyl blue”, MTT, Sigma Chemicals, cat. no. M 2128); and 0.1 mg/mlof N-methyl-dibenzopyrazine methyl sulfate salt (“phenazinmethosulfate”, PMS, Sigma Chemicals cat. no. P 9625). The gel wasincubated at room temperature in the dark until the coloured,blue-violet band was clearly visible (usually 5-90 minutes) and was thenrinsed in 10% acetic acid, 5% glycerol and air-dried.

The silver stained gel is shown in FIG. 2, lane 1. As it appears fromthe figure, numerous proteins were present at this step of thepurification. By enzyme staining, however, only the band marked with anarrow in FIG. 2 was stained. (results not shown).

1.7. Gel Filtration

This step in the purification was carried out on an FPLC system(Pharmacia) equipped with a XK26/70 column (2.6×66 cm, Pharmacia) with abed volume of 350 ml. The column was packed with Sephacryl S-200 HR(Pharmacia) according to the instructions of the manufacturer. Thebuffer was 20 mM Tris-Cl, 500 mM NaCl, pH 7.5 and the flow rate was 0.5ml/min. The UV-absorbance at 280 nm was recorded. Fractions of 2.5 mlwere collected with a FRAC-100 fraction collector (Pharmacia) which wasplaced in a refrigerator (4° C.) next to the FPLC. The concentratedpreparation of hexose oxidase was clarified by centrifugation at 30,000rpm in a SW60 swinging bucket rotor (Beckman) in an L7 ultracentrifuge(Beckman) for 60 min at 4° C. An aliquot of 3.0-4.0 ml of thesupernatant was mixed with 5% glycerol (Sigma Chemicals, cat. no. G7757), filtered through a disposable filter unit with 0.22 μm pore size(Millipore, cat. no. SLGV 025 BS) and applied onto the column using anSA-5 sample applicator (Pharmacia) connected to the inlet of the column.Fractions showing hexose oxidase activity were identified using theassay method described above (10 μl of sample, 15 min of incubationtime) and stored separately at −18° C. until further processing.

The UV-profile and the elution position of hexose oxidase is shown inFIG. 3. As it appears from this figure, a substantial amount ofUV-absorbing material was eliminated in this step. Electrophoreticanalysis by native PAGE and silver staining (FIG. 2, lane 2) showed thatonly a few contaminating components remained after this step.

1.8. Determination of Molecular Weight of Native Hexose Oxidase byAnalytical Gel Filtration

The molecular weight of native hexose oxidase was determined by gelfiltration on Sephacryl S-200 Superfine (Pharmacia). Column dimensions,buffer, flow rate and fraction collection were as described above. Bluedextran for determination of the void volume (v_(o)) of the column andthe following standard proteins for calibration of the column wereobtained from Pharmacia: Ovalbumin (43,000), albumin (67,000), catalase(158,000) and aldolase (252,000). A sample containing hexose oxidase wasobtained by DEAE-Sepharose chromatography as described above. Based ondetermination of the elution volumes (v_(e)) of the standard proteinsand of hexose oxidase, the corresponding K_(av) values(v_(e)−v_(o)/v_(t)−v_(o)) were calculated. Finally, the K_(av) values ofthe standard proteins were plotted against the corresponding log(molecular weight) values. The K_(av) of hexose oxidase corresponded toa native molecular weight of approximately 110,000. This is in goodagreement with Sullivan & Ikawa (1973), who found a molecular weight ofabout 130,000. Kerschensteiner & Klippenstein (1978) reported amolecular weight of 140,000, also as determined by gel filtration.

1.9. Cation Exchange Chromatography

This step was carried out on a SMART Micropurification ChromatographySystem (Pharmacia) equipped with a HR5/5 column (Pharmacia, 0.5×5 cm,bed volume 1.0 ml) packed with S-Sepharose Fast Flow (Pharmacia). Thecolumn was equilibrated in A-buffer: 50 mM sodium acetate, pH 4.5(prepared by adjusting 50 mM acetic acid to pH 4.5 with NaOH). Thebuffer B used for gradient elution contained 50 mM sodium acetate, 500mM NaCl, pH 4.5. Fractions from gel filtration were desalted onpre-packed, disposable Sephadex G-25 columns (PD-10, Pharmacia) whichwere equilibrated and eluted with 25 mM sodium acetate, pH 4.5. Twentyml of desalted sample derived from 6 gel filtration fractions with highhexose oxidase activity were applied onto the column from a 50 mlSuperloop (Pharmacia) at a flow rate of 250 μl/min. The column was thenwashed with 4 bed volumes of buffer A at the same flow rate. Boundproteins were eluted with a gradient from buffer A to buffer B over 5ml. Fractions of ²⁵⁰ μl were collected during gradient elution andassayed for hexose oxidase activity as described above (1 μl of sample,15 min of incubation time) and stored at −18° C. until further use.

The resultant preparation of hexose oxidase was analyzed by native PAGEand silver staining (FIG. 2, lane 3). The hexose oxidase band was nowthe only significant band, although small amounts of contaminatingproteins were also observed.

1.10. Analytical Sodium Dodecyl Sulphate PAGE (SDS-PAGE)

Fractions from S-Sepharose chromatography that showed hexose oxidaseactivity were also analyzed by SDS-PAGE according to Laemmli (1970).Minigels of 12.5% acrylamide/bisacrylamide (37.5:1 mixture) with athickness of 0.75 mm were run in a Mini-Protean II apparatus (Bio-Rad).The gels were stained with 0.1% Coomassie Brilliant Blue R-250, 10%acetic acid, 40% ethanol and destained in 10% acetic acid, 30% ethanol.

The result of the electrophoresis is shown in FIG. 4, lane 1. Thepurified preparation of hexose oxidase showed strong bands at relativemolecular weights of 40 kD and 29 kD, respectively and faint bands at 60kD and 25 kD, respectively. Furthermore, two sharp doublet bands at 55kD and 57 kD were observed.

1.11. SDS-PAGE Followed by Blotting and Staining for Carbohydrate

The presence of carbohydrate in the isolated hexose oxidase was examinedwith the DIG Glycan Detection Kit (Boehringer Mannheim), which isdesigned for detection of microgram amounts of sugars in glycoconjugateson blots. In principle, adjacent hydroxyl groups in carbohydrates areoxidized to aldehydes. Digoxigenin is then covalently bound to thealdehyde groups and subsequently detected with ananti-dig-oxigenin-alkaline phosphatase antibody conjugate.

Purified hexose oxidase from cation exchange chromatography was run on a12% SDS-PAGE gel as described above, blotted to nitrocellulose accordingto standard procedures and stained for carbohydrate with the GlycanDetection Kit according to the instructions of the manufacturer. None ofthe hexose oxidase bands at 60 kD, 40 kD, 29 kD and 25 kD were stained.Only the sharp doublet band at 57 kD-55 kD was intensely stained(results not shown). The 57 kD-55 kD doublet band was later identifiedas a residual contaminant as described below.

Thus, it could be concluded that none of the hexose oxidase componentsseen in SDS-PAGE were glycosylated.

1.12. Isoelectric Focusing

Hexose oxidase fractions from S-Sepharose chromatography were pooled andconcentrated by centrifugal ultrafiltration in Centricon concentrators(Amicon) and analyzed by isoelectric focusing (IEF) on Isogel agaroseplates, pH 3-10, according to the instructions of the manufacturer (FMCBioproducts, Rockland, Me., USA). A mixture of pI markers (FMCBioproducts) were run in parallel with the hexose oxidase samples. Themixture consisted of cytochrome C (pI=10.2), myoglobin major/minor band(7.4/7.0), carbonic anhydrase (6.1), β-lactoglobulin A/B (5.4/5.5),ovalbumin (4.8), glucose oxidase (4.2) and amyloglucosidase (3.6). Thegels were stained with Coomassie Brilliant Blue R-250. As shown in FIG.5, lane 1, the purified preparation of hexose oxidase was composed oftwo variants with pI's of 4.3 and 4.5, respectively. Purified hexoseoxidase was also analyzed by isoelectric focusing on pre-castpolyacrylamide gels, pH 3.5-9.5 (Pharmacia, Ampholine PAGplates)according to the instructions of the manufacturer. These gels werestained for enzyme activity by incubation in a staining mixture asdescribed above for native polyacrylamide gels. As shown in FIG. 5, lane2, both pI variants were enzymatically active.

1.13. Chromatofocusing

The observation of several bands in SDS-PAGE of hexose oxidase purifiedon S-Sepharose as the final step aroused the suspicion that one or moreof the bands might represent residual contaminants. Furthermore, theS-Sepharose chromatography consistently gave low recoveries. Therefore,chromatofocusing was introduced as a last purification step instead ofcation exchange chromatography on S-Sepharose.

Chromatofocusing was carried out on the SMART chromatography systemequipped with a Mono P HR 5/5 column (0.5×5 cm, Pharmacia) with a bedvolume of 1 ml and a 50 ml Superloop for sample application. The startbuffer for separation in the interval between pH 5.0 and 3.5 was 25 mMpiperazine adjusted to pH 5.5 with HCl. The eluent was Polybuffer 74(Pharmacia) 10-fold diluted with water and adjusted to pH 3.5 with HCl.The column was pre-treated and equilibrated with start buffer asrecommended by the manufacturer.

Sample preparation was carried out in the following manner: In a typicalexperiment the best fractions from two gel filtration runs (2×4fractions, 20 ml) were pooled and passed through a column of 1 ml ofPhenyl Sepharose 6 Fast Flow (high sub, Pharmacia) which had been packedin a disposable Poly-prep column (Bio-Rad) and equilibrated in thebuffer used for gel filtration (20 mM Tris-Cl, 500 mM NaCl, pH 7.5).This treatment almost completely removed remaining amounts of the redprotein phycoerythrin and other coloured substances which were adsorbedto the gel matrix at this ionic strength, and thereby eliminatedcontaminants that were only partially removed during the other steps ofthe purification process. The Phenyl Sepharose column was discardedafter use. Hexose oxidase activity was quantitatively recovered in theeffluent which was then desalted on pre-packed disposable Sephadex G-25columns (PD-10, Pharmacia) equilibrated and eluted with start buffer.

Before sample application 1 ml of eluent was pumped onto the column. Theflow rate was 0.5 ml/min. After sample application the pH gradient wasformed by pumping 11 ml of eluent through the column. During the pHgradient elution 44 fractions of 250 μl were collected. Fractionscontaining hexose oxidase were identified by the assay method describedabove (1 μl of sample, 15 min of incubation time) and stored at −18° C.until further use.

Hexose oxidase purified by chromatofocusing was analyzed by native PAGEand silver staining (FIG. 2, lane 4) and by SDS-PAGE and staining withCoomassie Brilliant Blue (FIG. 4, lane 2). In native PAGE the hexoseoxidase band was the only significant band, and only very low amounts ofcontaminants were observed. By SDS-PAGE it was clearly demonstrated thatthis purification method was able to remove the sharp doublet band at 57kD and 55 kD. The band at 25 kD observed after S-Sepharosechromatography was very faint after chromatofocusing.

In conclusion, hexose oxidase obtained by DEAE chromatography, gelfiltration and chromatofocusing showed one band in native PAGE. InSDS-PAGE strong bands at 40 kD and 29 kD and a weak band at 60 kD wereobserved.

Since the intensity of the 60 kD component, relative to the 40 kD and 29kD components, varied between different preparations of the enzyme, itwas hypothesized that the 29 kD and 40 kD polypeptides might originatefrom proteolytic processing of an about 60 kD precursor. This would fitwith the idea of a homo-dimeric structure of the enzyme with a nativemolecular weight of 110,000-120,000 as it was actually found by gelfiltration, as described above. Furthermore, this hypothesis would beconsistent with the results obtained by Kerschensteiner and Klippensteinwho found a native molecular weight of 140,000 in gel filtration and asubunit molecular weight of 70,800 in SDS-PAGE (Kerschensteiner andKlippenstein, 1978).

EXAMPLE 2

Generation and Amino Acid Sequence Analysis of Peptide Fragments ofHexose Oxidase

2.1. Digestion of Purified Hexose Oxidase with Cyanogen Bromide

This procedure was carried out while cation exchange chromatography onS-Sepharose was still used as the last purification step.

Hexose oxidase obtained by purification on DEAE Sepharose, SephacrylS-200, and S-Sepharose was transferred to a volatile buffer bybuffer-exchange on a pre-packed PC3.2/10 Fast Desalting Columncontaining Sephadex G-25 Superfine (Pharmacia, 0.32×10 cm, bed volume0.8 ml) which was mounted in the above SMART system. The column wasequilibrated and eluted with 200 mM ammonium bicarbonate (BDH, AnalaR).To obtain a satisfactory recovery it was necessary to add 500 mM sodiumchloride to the hexose oxidase sample before injection.

Eluted, buffer-exchanged hexose oxidase was distributed into 1.5 mlmicrocentrifuge tubes and lyophilized in a Speedvac concentrator (SavantInstruments). Cyanogen bromide (CNBr, Pierce), 200 μl of a 10 mg/mlsolution in 70% v/v formic acid (Promega), was added. (Reagents fromPromega were components of a “Probe Design™ Peptide Separation System”cat. no. V6030). The tubes were incubated overnight in the dark at roomtemperature. The solutions were then dried in the speedvac concentrator,resuspended in 50 μl of water and re-dried.

2.2. Separation of Cyanogen Bromide Fragments by High ResolutionSDS-PAGE and Electroblotting to Polyvinylidene Difluoride (PVDF)Membrane

The peptides generated by cyanogen bromide digestion were separated byhigh resolution SDS-PAGE according to Schägger & von Jagow (1987). Thissystem provides excellent separation of low molecular weight peptides(20-250 amino acid residues). The gel system consisted of a 16.5%separation gel, a 10% spacer gel and a 4% stacking gel, all made using a29:1 acrylamide/bisacrylamide mixture from Promega.

Minigels with a thickness of 0.75 mm were run in a Mini-Protean IIapparatus (Bio-Rad). Ammonium persulfate andN,N,N′,N′-tetramethyl-ethylenediamine (TEMED) were from Bio-Rad. SDS wasfrom United States Biochemical (ultrapure). Tris was from Fluka (cat.no. 93350). Tricin and sodium thioglycate were from Promega. Glycin(p.a.), 2-mercaptoethanol (p.a.) and bromophenol blue was from Merck andglycerol from GIBCO BRL (ultrapure). Sodium thioglycolate, 0.1 mM, wasadded to the cathode buffer just before use to prevent chemical blockageof the amino-termini of the peptides during the separation. The gel waspre-run for 60 min at 30 V to allow the thioglycolate to scavenge anyamino-reactive substances.

Sample preparation: The dried cyanogen bromide peptide fragments wereresuspended in 30 μl of gel loading buffer containing 63 mM Tris-Cl, pH6.8, 1% SDS, 2.5% 2-mercaptoethanol, 10% glycerol and 0.0012%bromophenol blue. Samples that turned yellow upon mixing, due to thecontent of residual formic acid were neutralized by addition of 1-3 μlof 1.0 M Tris base until the blue colour was restored. The samples weredenatured by heating at 95° C. for 5 min before application on the gel.A mixture of Low-Range Protein molecular weight standards (Promega) withmolecular weights between 31,000 and 2,500 were run in parallel with thehexose oxidase peptide samples. The electrophoresis was run at 150Vconstant voltage.

Electrophoretic transfer to PVDF membrane was carried out in a MiniTrans-Blot Electrophoretic Transfer Cell (Bio-Rad) according to theinstructions of the manufacturer. Three sheets of Problott membrane(Applied Biosystems) cut to the size of the gel were wetted briefly inmethanol (Merck, p.a) and then soaked in transfer buffer (25 mM Tris,192 mM glycine, pH 8.5, pre-cooled to 4° C.) until assembly of theblotting sandwich. After electrophoresis the gel was incubated intransfer buffer for 5 min at 4° C. and then assembled into a transfersandwich having the following layers: A sheet of Whatman paper (3 MMchr), two sheets of Problott membrane, the SDS-PAGE peptide separationgel, the third sheet of Problott, and a final sheet of Whatman paper.The sandwich was oriented with the two sheets of Problott membranetoward the positive electrode in the electrode assembly. The coolingunit was mounted in the buffer chamber before it was filled withpre-cooled transfer buffer, and the transfer was then performed at roomtemperature for 60 min at 100V constant voltage. During transfer thecurrent increased from about 270 mA to about 400 mA.

After transfer the membrane was washed in water for 1 min and thenstained for 30-45 sec in 100 ml of freshly prepared staining solutioncontaining 0.1% Coomassie Brilliant Blue R-250 (Bio-Rad), 5% acetic acid(Merck, p.a) and 45% methanol (Merck, p.a). The membrane was thendestained with 3 changes of about 80 ml of freshly prepared 5% aceticacid, 45% methanol for 30-60 sec each. The membrane was finally washedin 3 changes of water to remove residual glycine and then air-dried.Well-resolved and relatively abundant bands of molecular weights ofabout 2.5 kD, 9 kD and 16 kD, respectively were excised and submitted toamino acid analysis and sequence analysis.

2.3. Amino Acid Analysis and Sequencing of a 9 kD Cyanogen BromideFragment of Hexose Oxidase

Amino acid analysis was carried out by ion exchange chromatography andpost-column derivatization with o-phtaldialde-hyde. Samples werehydrolyzed at 110° C. for 20 h in 6 M HCl, 0.05% phenol and 0.05%dithiodipropionic acid (Barkholt and Jensen, 1989). Peptides weresequenced on an automated protein/peptide sequencer from AppliedBiosystems, model 477A, equipped with on-line PTH analyzer, model 120Aand data analysis system. Protein sequencing reagents were obtained fromApplied Biosystems. Amino acid analysis and peptide sequence analysiswas kindly performed by Arne L. Jensen, Department of Protein Chemistry,University of Copenhagen, Denmark.

The peptide sequence identified by analysis of the 9 kD fragment isshown in Table 2.1.

The initial yield of phenylthiohydantoin-tyrosine (PTH-Tyr) at step onewas 22 pmol. The amino acid composition of the 9K fragment is shown inTable 2.2.

TABLE 2.1 Peptide sequence obtained by sequence analysis of a 9 kDcyanogen bromide fragment of hexose oxidase Origin of sequenced SequenceAmino acid peptide identification sequence HOX, 9K CNBr HOX-1 peptideY-E-P-Y-G-G-V-P- fragment Abbreviations: Y = Tyr; E = Glu; P = Pro; G =Gly; V = Val

TABLE 2.2 Amino acid composition of a 9 kD cyanogen bromide fragment ofhexose oxidase Amino Acid mol % N Asx 16.4 14 Thr 4.8 4 Ser 4.6 4 Glx9.9 8 Pro 8.1 7 Gly 11.2 9 Ala 4.3 4 Cys 0 0 Val 5.2 5 Met 0.2 0 Ile 3.63 Leu 9.3 8 Tyr 6.1 5 Phe 4.6 4 His 1.0 1 Lys 8.1 7 Arg 2.7 2 Trp ND¹⁾ —total 100.0 85 ¹⁾Not determined2.4. Preparative SDS-PAGE and Electroblotting to PVDF Membrane

The following procedure was carried out in order to obtain amino acidsequences which were specifically known to stem from either the 40 kD orthe 29 kD polypeptide of the hexose oxidase preparation.

Preparative SDS-PAGE gels were run according to Laemmli (Laemmli, U.K.,1970). Minigels containing 12.5% acrylamide/bisacrylamide (37.5:1mixture) with a thickness of 0.75 mm were run in a Mini-Protean IIapparatus (Bio-Rad). The solution of acrylamide (BDH, cat. no. 44313)and N,N′-methyl-ene-bis-acrylamide (BDH, cat. no. 44300) was stored overmixed bed ion exchange resin (Bio-Rad, cat. no. 142-6425). The sourcesof all other reagents were as described above.

Sample preparation: Fractions from chromatofocusing were concentrated bycentrifugal ultrafiltration at 4° C. in Ultra-free-MC filter units withNMWL 10,000 and a sample capacity of 400 μl (Millipore, cat. no. UFC3LGC25). The retentate was mixed with one volume of gel loading 2× buffercontaining 125 mM Tris-Cl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 20%glycerol and 0.0025% bromophenol blue. Samples that turned yellow uponmixing, due to the content of acidic Polybuffer components, wereneutralized by addition of 1-3 μl of 1.0 M Tris base until the bluecolour was restored. The samples were denatured by heating at 95° C. for5 min and applied on the gel in aliquots of about 30 μl per lane.

A mixture of molecular weight marker proteins (Bio-Rad) with molecularweights ranging from 97,400 to 14,400 was run in parallel with thehexose oxidase samples. The electrophoresis was run at low current, 10mA per gel, in order to minimize the risk of thermally induced, chemicalmodification of the sample proteins.

Electrophoretic transfer to PVDF membrane was carried out as describedabove, except that one sheet of Immobilon P Membrane (Millipore, cat.no. IPVH 15150) was used instead of three sheets of Problott. Thesandwich was oriented with the blotting membrane toward the positiveelectrode in the electrode assembly.

After transfer the Immobilon P membrane was rinsed in water for 10 secand then stained for 45-60 sec in 100 ml of freshly prepared stainingsolution containing 0.025% Coomassie Brilliant Blue R-250, 5% aceticacid and 40% methanol. The membrane was then destained for 2-3 min in250 ml of freshly prepared 5% acetic acid, 30% ethanol (96% v/v,Danisco, Denmark). The membrane was finally air-dried and stored at 4°C.

The band pattern on the blot was identical to the pattern seen inanalytical SDS-PAGE after final purification by chromatofocusing. Itshowed strong bands at 40 kD and 29 kD, in addition to a faint band at60 kD.

Bands at 40 kD and 29 kD were excised from the blot and used for aminoacid analysis and for enzymatic digestion of polypeptides bound to themembrane, as described below. The amount of 60K material was too low topermit any further analysis of this polypeptide.

2.5 Amino Acid Analysis of 40 kD and 29 kD Polypeptides of HexoseOxidase

The amino acid compositions of the 40 kD and 29 kD components of hexoseoxidase are shown in Table 2.3.

TABLE 2.3 Amino acid composition of 40 kD and 29 kD polypeptides ofhexose oxidase mol % Amino acid 40K 29K Asx 11.5 12.5 Thr 5.9 5.2 Ser6.1 4.7 Glu 9.7 15.1 Pro 5.2 5.4 Gly 13.6 9.7 Ala 6.6 6.4 Cys 1.1 0.9Val 7.3 5.5 Met 1.5 2.3 Ile 3.7 4.4 Leu 8.5 8.6 Tyr 4.2 5.3 Phe 5.5 4.1His 2.2 1.4 Lys 3.9 6.1 Arg 3.5 2.4 Trp ND¹⁾ ND¹⁾ Total 100.0 100.0¹⁾Not determined2.6. Enzymatic Digestion of PVDF-Bound Hexose Oxidase Polypeptides

Digestion of hexose oxidase polypeptides bound to PVDF and extraction ofthe resultant proteolytic peptides was performed as described byFernandez et al. (1992) and Fernandez et al. (1994).

Digestion of the 40 kD polypeptide of hexose oxidase: Eleven 40K bandswith an estimated total protein content of about 5 μg (corresponding toabout 125 pmol) were excised from the Coomassie blue-stained PVDFmembrane, destained in methanol for 1-2 min and rinsed in water for 2-3min. The membrane bands were then diced into 1×1 mm pieces andtransferred to microcentrifuge tubes. A blank region of PVDF membraneserved as a background control. The diced membrane pieces were soaked in50 μl of digestion buffer containing 1% (v/v) hydrogenated Triton X-100(RTX-100, Sigma Chemicals, cat. no. X-100R-PC, or Calbiochem, proteingrade, cat. no. 648464), 10% acetonitrile (Merck, Gradient GradeLichrosolv) and 100 mM Tris-Cl, pH 8.0. The proteolytic enzyme selectedfor the digestion was endoproteinase Lys-C (endoLys-C) which cleavespeptide chains at the C-terminal side of lysine residues. An aliquot of5 μg of endoLys-C (Boehringer Mannheim, sequencing grade, cat. no. 1047825) was reconstituted by addition of 20 μl of water. Two μl of enzymesolution, corresponding to 0.5 μg was added (enzyme:substrate ratio1:10). Digestion was carried out at 37° C. for 22-24 h.

After digestion the samples were sonicated in an ultrasonic tank (Elmatransonic) for 5 min and centrifuged at 1700 rpm in a microcentrifugefor 5 min, and the supernatant was then transferred to a new tube.Consecutive washes with 50 μl of digestion buffer and 100 μl of 0.1%trifluoroacetic acid (TFA, Pierce, cat. no. 28902) were performed withsonication and centrifugation as described above. All supernatants werepooled, resulting in an extract volume of 200 μl. The extracts were keptat −18° C. until peptide purification.

Digestion of the 29 kD polypeptide of hexose oxidase was performed asdescribed for the 40 kD component, except that four bands with a totalprotein content of 2.4 μg (about 80 pmol), according to amino acidanalysis, were used.

2.7. Purification of EndoLys-C Generated Peptides

The peptide fragments obtained by digestion of 40 kD and 29 kDpolypeptides of hexose oxidase were separated on the SMARTchromatography system. The system was equipped with avariable-wavelength μPeak monitor and a fraction collector bowl for 60vials. The reversed phase column used for the separation was asilica-based μRPC C2/C18 SC2.1/10 narrow-bore column (Pharmacia, columndimensions 2.1×100 mm, particle size 3 μm, average pore size 125 Å). Thebuffers were A: 0.1% TFA (Pierce) in Milli-Q water and B: 0.1% TFA inacetonitrile (Merck, gradient grade Lichrosolv). The buffers werefiltered and degassed by vacuum filtration on a 0.5 μm fluoropore filter(Millipore, cat. no. FHLP04700). The flow rate was 100 μl/min.UV-absorbance in the effluent was monitored at 220 nm, 254 nm and 280nm. The gradient was 0-30% B (0-65 min), 30-60% B (65-95 min) and 60-80%B (95-105 min). The column was then washed at 80% B for 10 min at 100μl/min and re-equilibrated in A-buffer for 45 min at 200 μl/min.Fractions of 50 μl were collected between t=15 min and t=105 min (3×60fractions) and stored at −18° C. until amino acid sequence analysis.

The peptide map obtained after endoLys-C digestion of the 40 kDpolypeptide is shown in FIG. 6. As seen in this figure, the digestionand HPLC separation resulted in several well-resolved peaks with a highsignal-to-noise ratio. A corresponding chromatogram of a blank digestionmixture (not shown) indicated that the peaks eluting later than t=83 minwere non-peptide, reagent-derived peaks, possibly UV-absorbingcontaminants of the hydrogenated Triton X-100 or residual traces ofCoomassie dye. The peaks labelled 1-5 in FIG. 6 were selected for aminoacid sequencing by the following criteria: 1) Peak height. 2) Apparentpurity. 3) High A₂₈₀:A₂₂₀ and/or high A₂₅₄:A₂₂₀ ratio indicating thepresence of aromatic amino acid residues, which are most useful forselection of PCR primer sequences due to their low genetic codedegeneracy. 4) Late elution time, which may indicate a relatively longpeptide.

The chromatogram of the 29 kD endoLys-C peptides is shown in FIG. 7.Obviously, this hexose oxidase component gave rise to only a fewsignificant peptide fragments compared to the 40 kD component in FIG. 6.When comparing the chromatograms, there was no indication of any peptidefragment being present in both digests. This finding suggests that the40K and 29K hexose oxidase components do not have amino acid sequencesin common, which would have been the case if the 29 kD chain wasgenerated by proteolytic conversion of the 40 kD polypeptide. (Comparedto the 40 kD digest, the 29 kD digest contained only small amounts ofthe contaminating substances eluting later than t=83 min. The reason forthis might be that hydrogenated Triton X-100 from Calbiochem was usedfor the 29 kD digestion, whereas the 40 kD digestion was carried outwith Triton X-100 from Sigma Chemicals).

Fractions corresponding to the peaks labelled 1 and 2 in the 29 kDpeptide map (FIG. 7) were subjected to amino acid sequencing.

2.8 Amino Acid Sequence Analysis of Proteolytically Generated Peptidesof Hexose Oxidase

The peptide sequences identified by analysis of fractions correspondingto peaks 1-5 in FIG. 6 (HOX-2, HOX-3, HOX-4, HOX-5 and HOX-6 peptides)and peaks 1-2 in FIG. 7 (HOX-7 and HOX-8 peptides) are shown in thebelow Table 2.4. The initial yields of PTH amino acids ranged from 46pmol of PTH-Tyr at step one in the HOX-5 peptide to 6 pmol of PTH-Ile atstep two in the HOX-8 peptide. As expected from the absorbances at 254nm and 280 nm, respectively of the selected peaks all the sequencedpeptides contained at least one aromatic amino acid residue.

TABLE 2.4 Peptide sequences obtained by sequence analysis ofendoproteinase Lys-C peptides derived from 40 kD and 29 kD polypeptidesof hexose oxidase Origin of Sequence sequenced identifi- peptide cationAmino acid sequence 40 K, HOX-2 A-I-I-N-V-T-G-L-V-E-S-G-Y-D- peak 1peptide X¹⁾-X²⁾-X³⁾-G-Y-X-V-S-S- 40 K, HOX-3D-L-P-M-S-P-R-G-V-I-A-S-N-L-W- peak 2 peptide F- 40 K, HOX-4D-S-E-G-N-D-G-E-L-F-X-A-(H)-T- peak 3 peptide 40 K, HOX-5 Y-Y-F-K peak 4peptide 40 K, HOX-6 D-P-G-Y-I-V-I-D-V-N-A-G-T-P-D- peak 5 peptide 29 K,HOX-7 L-Q-Y-Q-T-Y-W-Q-(E)-(E)-(D)- peak 1 peptide 29 K, HOX-8X-I-(R)-D-F-Y-E-E-M- peak 2 peptide

Tentatively identified residues are shown in parentheses.

-   1) Residue no. 15 was identified as either Asp or Asn.-   2) residue no. 16 was identified as either Asp or Ala.-   3) Residue no. 17 was identified as either Arg or Trp.-   HOX-2 peptide=SEQ ID NO:9-   HOX-3 peptide=SEQ ID NO:10-   HOX-4 peptide=SEQ ID NO:11-   HOX-5 peptide=SEQ ID NO:12-   HOX-6 peptide=SEQ ID NO:13-   HOX-7 peptide=SEQ ID NO:14-   HOX-8 peptide=SEQ ID NO:15

EXAMPLE 3

Isolation of Hexose Oxidase Gene from Chondrus crispus

3.1. Purification of RNA from Chondrus crispus

Freshly collected fronds of Chondrus crispus were rinsed with cold waterand immediately stored in liquid nitrogen until further use. About 15grams of Chondrus crispus thallus frozen in liquid nitrogen washomogenized to a fine powder in a mortar. The frozen, homogenizedmaterial was transferred to a 50 ml tube (Nunc, cat. no. 339497)containing 15 ml extraction buffer (8M guanidinium hydrochloride; 20 mM2-(N-morpho-lino)ethanesulfonic acid (MES), pH 7.0; 20 mMethylenedi-aminetetraacetic acid (EDTA); 50 mM β-mercaptoethanol).

The tube was vortexed and kept cold (0° C.) during the following stepsunless other temperatures are indicated. Then the tube was centrifugedfor 20 minutes at 6,000×g in a Heraeus Omnifuge 2.0RS and theRNA-containing supernatant (about 15 ml) was carefully collected andtransferred to a pre-chilled 50 ml tube. 1.5 ml 2 M sodium acetate, pH4.25, 15 ml water saturated phenol and 3 ml chloroform:isoamyl alcohol(49:1) was added to the tube containing the RNA extract.

The tube was subsequently vortexed vigorously for ½ minute and thephases were separated by centrifuging the tube for 20 minutes in anOmnifuge at 6,000×g. The aqueous phase (about 17 ml) was transferred toa 30 ml Corex tube (Sorvall, cat. no. 00156) and an equal volume (i.e.about 17 ml) of cold isopropanol was added. The tube was vortexed againand incubated for at least 1 hour at −20° C. The precipitated RNA waspelleted by centrifugation for 20 minutes at 10,000 rpm using a SorvallRC-5B centrifuge provided with a pre-chilled SS24 rotor. The supernatantwas discarded and the pelleted RNA was resuspended in 4 ml 0.3 M sodiumacetate, pH 5.5 and 12 ml 96% ethanol was added.

The Corex tube was then vortexed and incubated again for at least 1 hourat −20° C. followed by a second pelleting of RNA by centrifugation for20 minutes as described above. The supernatant was carefully discardedand the RNA pellet resuspended in 2 ml 0.15 M sodium acetate, pH 5.5.Then 8 ml 4 M sodium acetate, pH 5.5. was added and the RNA wasprecipitated on ice for 30 minutes and pelleted again as describedabove. The RNA pellet was washed in 70% ethanol and resuspended in 500μl water. The resuspended RNA was transferred to a microcentrifuge tubeand stored at −20° C. until further use.

The purity and concentration of the RNA was analyzed by agarose gelelectrophoresis and by absorption measurements at 260 nm and 280 nm asdescribed in Sambrook et al. (1989).

3.2. Isolation of Poly-Adenylated RNA from Chondrus crispus

Poly-adenylated RNA was isolated from total RNA using magnetic beadscontaining oligo dT (Dynabeads® Oligo (dT)₂₅, in mRNA Purification Kit™,Dynal). Approximately 100 μg total RNA was mixed with 1 mg Dynabeads®Oligo (dT)₂₅ and poly-adenylated RNA was isolated as described in theprotocol for the mRNA Purification Kit™. The yield of poly-adenylatedRNA isolated with Dynabeads® was between 1 and 3%.

Other methods were used in the isolation of poly-adenylated RNA fromChondrus crispus including using columns packed witholigo-(dT)-cellulose (Clontech, cat. no. 8832-2) or pre-packed columns(mRNA Separator Kit™, Clontech, cat. no. K1040-1) as described in theprotocol for the mRNA Separator Kit™. The yield of poly-adenylated RNAisolated on oligo-(dT) columns was between 0.1 and 1% of the initialtotal RNA. Poly-adenylated RNA isolated on oligo-(dT) columns was usedin cDNA synthesis reactions as described below (3.4), but the yield offirst strand cDNA was very low (less than 1%).

The reason for the lower yield and the poorer performance of RNAisolated on oligo-(dT) columns compared to Dynabeads® purified RNA couldbe the presence of carbohydrates or proteoglycans in the extract oftotal RNA. Carbohydrates contaminating the total RNA preparations havebeen shown to impede the purification of poly-adenylated RNA and toinhibit cDNA synthesis and therefore, methods for the purification ofRNA free of carbohydrates have been developed (Groppe et al., 1993; Yehet al.) However, poly-adenylated RNA purified with these methods was notas effective in cDNA synthesis reactions as RNA isolated withDynabeads®. Accordingly, poly-adenylated RNA purified using Dynabeads®was used as template in first strand cDNA synthesis reactions (cf. 3.4below).

3.3. Hexose Oxidase Specific Oligonucleotides

Synthetic oligonucleotides were synthesized (DNA technology, ApS,Forskerparken, DK-8000 Aarhus C, Denmark) based on the amino acidsequences derived from hexose oxidase peptides HOX-2, HOX-3 and HOX-4(Table 2.4). Table 3.1 shows the oligonucleotides and theircorresponding amino acid sequences. Also shown in Table 3.1 is the DNAsequence of the primers used in DNA sequencing or in PCR.

TABLE 3.1 Nucleotide sequences of synthetic oligonucleotides specificfor hexose oxidase Hox- Hox- peptide primer Hox-2  A   I   I   N   V   T   G  L   V   E   S   G   Y   D   X   X   X   G   Y   X   V   S   S Hox2-3+^(5′)YTI GTI GAR WSI GGN TAY GA^(3′) Hox-3  D   L   P   M   S   P   R   G       V   I   A   S   N   L   W   FHox3-2−    ^(5′)CAN TAD CGN AGI TTR RAI ACC AA^(5′) Hox-4  D   S   E   G   N   D   G   E   L   F   X   A   H   T Hox4-1+       ^(5′)GAR GGI AAY GAY GGI GAR CTN TT^(3′) Hox4-2−        ^(3′)CTYCCN TTR CTR CCI CTY GAI AA^(5′) Hox5+    ^(5′)ATT GGG GCT CCT TCA AGACCT T^(3′) Hox5−    ^(5′)TGA TGA TTC CAA AGT TTC^(3′) Hox6+    ^(5′)TTGGAA GAA TAC GGT TGG^(3′) Hox7−    ^(5′)TAC TAT TTC GTC TGC TTG GG^(3′)Hox8−    ^(5′)GAA CTC TTC CGT GGT CTC CT^(3′) Hox10−    ^(5′)CCA CCT GCGTGT TGG GGT CT^(3′) Hox11+    ^(5′)CAG ATC TAC AAA ACA TGC GAG^(3′)Hox12−    ^(5′)TGT CGC AGA CTG TAC TTG^(3′) Hox13−    ^(5′)GAG TGT ACACGA CAT AAA^(3′) Hox5′-1    ^(5′)ATG GCT ACT CTT CCC CAG AAA G^(3′)

When Y is C or T, R is A or G; when W is A or T, S is C or G; when D isA, G or T, N is A, C, G or T, and I=deoxy Inosine.

-   Hox-2=SEQ ID NO:9-   Hox2-3+=SEQ ID NO:16-   Hox-3=SEQ ID NO:10-   Hox3-2−=SEQ ID NO:17-   Hox-4=SEQ ID NO:11-   Hox4-1+=SEQ ID NO:18-   Hox4-2−=SEQ ID NO:19-   Hox5+=SEQ ID NO:20-   Hox5−=SEQ ID NO:21-   Hox6+=SEQ ID NO:22-   Hox7−=SEQ ID NO:23-   Hox8−=SEQ ID NO:24-   Hox10−=SEQ ID NO:25-   Hox11+=SEQ ID NO:26-   Hox12−=SEQ ID NO:27-   Hox13−=SEQ ID NO:28-   Hox5′-1=SEQ ID NO:29    3.4 cDNA Synthesis and Polymerase Chain Reaction (PCR)

Poly-adenylated RNA was used as template in first strand cDNA synthesisreactions with commercially available kits. About 1 μg poly-adenylatedRNA was reverse transcribed as described in the protocol for Maraton™cDNA Amplification Kit (Clontech, cat. no. K1802-1) with Hox3-2− orHox4-2− as primers. In the subsequent PCR amplification the anchor oradaptor primer of the kit was used in addition to the hexose oxidasespecific primers Hox3-2− or Hox4-2−, respectively. The buffers used andthe conditions for amplification was essentially as described in theprotocol for the Maraton™ cDNA Amplification Kit. PCR amplification wascarried out with AmpliTaq (Perkin-Elmer Cetus) using a Perkin-ElmerThermalcycler 480™ programmed to 30 cycles at 1 min at 94° C., 2 min at55° C. and 2 min at 72° C. Gel electrophoresis of 5 μl of the reactionmixture in a 1% agarose gel (SeaPlaque® GTG, FMC) showed DNA fragmentswith approximate sizes of 600 base pairs (bp) with primer Hox4-2− and of700 bp with primer Hox3-2−.

These DNA fragments were purified from the agarose gel using acommercially available kit (QIAEX™ Gel Extraction Kit, cat. no. 20020,QIAGEN) and about 100 ng fragment was ligated to 50 ng plasmid pT7 Blueas described in the protocol for pT7 Blue T-Vector Kit (Novagen, cat.no. 69829-1). Escherichia coli DH5α (Life Technologies, cat. no.530-8258SA) or E. coli NovaBlue (Novagen) was transformed with theligation mixture, and white, recombinant colonies were analyzed further.

Plasmid DNA from such colonies was purified using QIAGEN Plasmid MidiKit (QIAGEN, cat. no. 12143) and subjected to DNA sequence analysisusing Sequenase (Sequenase Version 2.0 DNA Sequencing Kit, USB). DNAsequencing reactions were subjected to acrylamide gel electrophoresis(Sequencing Gel-Mix® 6, Life Technologies). DNA sequence analysis of the700 bp fragment showed an open reading frame with a coding capacity of234 amino acids.

Table 3.2. below shows that all the peptide sequences from the 40 kDpolypeptide, i.e. HOX-2, HOX-3, HOX-4, HOX-5, and HOX-6, were found inthe 234 amino acid sequence derived from this open reading frame. Thus,it was concluded that the 700 bp fragment encoded part of the hexoseoxidase gene. The DNA sequence of the 600 bp fragment was shown to beidentical to the proximal 600 bp of the 700 bp fragment (see Table3.2.).

Primers Hox2-3+ and Hox3-2− were used similarly in cDNA synthesis andPCR amplification experiments. About 50 ng poly-adenylated RNA wasreverse transcribed with Hox3-2− as primer as described in the protocolfor 3′-Amplifinder™ RACE Kit (Clontech, cat. no. K1801-1). In thesubsequent PCR amplification primers Hox2-3+ and Hox3-2− were used. Thebuffers used and the conditions for amplification were essentially asdescribed for AmpliTaq polymerase (Perkin-Elmer Cetus) and in theprotocol for 3′-Amplifinder™ RACE Kit. Gel electrophoresis of 5 μl ofthe PCR amplification mixture showed a fragment with a size of 407 bp.

This fragment was purified, inserted into plasmid pT7 Blue and sequencedas described above. The DNA sequence of this fragment was shown to beidentical to the distal 407 bp of the 700 bp fragment.

The DNA sequence downstream of the 700 and 407 bp fragments wasamplified with the 3′Amplifinder™ RACE Kit (Clontech) using the anchorprimer of the kit as 3′primer and the hexose oxidase specific primersHox5+ and Hox4+ as gene specific 5′ primers. The buffers and theconditions for amplification were as described above. PCR and analysisof the reaction mixture on agarose gels showed a fragment with the sizeof about 1.3 kb. The fragment was isolated and subjected to DNA sequenceanalysis as described above. The DNA sequence of this 1.3 kb fragmentshowed an open reading frame of 357 amino acids. This 357 amino acidreading frame contained the amino acid sequences of the peptides HOX-1,HOX-3, HOX-4, HOX-5, HOX-7 and HOX-8. Therefore, it was concluded thatthe 1.3 kb DNA fragment encoded the 9 kD CNBr fragment, the 29 kDpolypeptide, and part of the 40 kD polypeptide of hexose oxidase.

A primer specific for the 5′ end of hexose oxidase, Hox5′-1, was usedtogether with an oligo-(dT) primer to amplify the assumed entire hexoseoxidase open reading frame. The gene was amplified using PCR, insertedinto pT7 Blue and sequenced as described above. The DNA sequence of this1.8 kb fragment was identical to the DNA sequences of the fragmentsdescribed above with minor differences. Since these differences could becaused by misincorporations during PCR amplifications, the entire hexoseoxidase gene was amplified and isolated from at least three independentPCR amplifications. Therefore, the DNA sequence presented in the belowTable 3.2. is composed of at least three independently derived DNAsequences in order to exclude PCR errors in the sequence.

The amino acid sequence derived from the open reading frame on the above1.8 kb DNA sequence is shown to contain all of the above HOX peptides,ie HOX-1 to HOX-8. Accordingly, the 1.8 kb DNA sequence codes for theabove 9 kD, 29 kD and 40 kD Chondrus crispus-derived hexose oxidasefragments. The molecular weight of this derived open reading framepolypeptide is consistent with the assumption that the polypeptide is asubunit (possibly a monomeric fragment) of a dimeric hexose oxidaseenzyme molecule.

3.5 Northern Blot Analysis of Chondrus crispus RNA

Total RNA isolated from Chondrus crispus was subjected to Northern blotanalysis. RNA was purified as described above (3.1) and fractionated ona denaturing formaldehyde agarose gel and blotted onto a HybondC filter(Amersham) as described by Sambrook et al. (1989). Using the primersHox2-3+ and Hox3-2− a 400 bp DNA fragment was synthesized by PCR asdescribed above (3.4). This fragment was purified from a 1.2% agarosegel (SeaPlaque® GTG, FMC) and labelled with ³²p as described by Sambrooket al. (supra). This radioactive hexose oxidase specific hybridizationprobe was used to probe the Northern blot. The conditions forhybridization was:

3.5.1. Prehybridization at 65° C. for two hours in a buffer containing10× Denhardt's solution (0.1% Ficoll, 0.1% poly-vinylpyrrolidone, 0.1%bovine serum albumin), 2×SSC (1×SSC is 0.15 M sodium chloride, 0.015 Msodium citrate, pH 7.0), 0.1% sodium dodecyl sulfate (SDS), and 50 μg/mldenatured salmon sperm DNA.

3.5.2. Hybridization at 65° C. for at least 14 hours in a buffercontaining 1× Denhardt's solution, 2×SSC, 0.1% dextran sulfate, 50 μg/mldenatured salmon sperm DNA, and ³²P labelled probe (approximately 10⁶dpm/ml). The filter was washed twice at 65° C. for 10 minutes in 2×SSC,0.1% SDS followed by two washes at 65° C. for 10 min in 1×SSC, 0.1% SDS.After the final wash for 10 minutes at 65° C. in 0.2×SSC, 0.1% SDS, thefilter was wrapped in Saran Wrap and exposed to an X-ray film (KodakXAR2) for two days at −80° C. using a Siemens-Titan HS intensifyingscreen. The resultant autoradiogram (FIG. 8) shows that a band with theapproximate size of 2 kb lighted up.

TABLE 3.2 Nucleotide sequence of 1.8 kb DNA sequence (SEQ ID NO:30) andthe open reading frame for a hexose oxidase amino acid sequence of 546amino acids derived from the DNA seqence (SEQ ID NO:31) TGAATTCGTGGGTCGAAGAG CCCTTTGCCT CGTCTCTCTG GTACCGTGTA TGTCAAAGGT 60 TCGCTTGCACACTGAACTTC ACG ATG GCT ACT CTT CCT CAG AAA GAC CCC 110                          Met Ala Thr Leu Pro Gln Lys Asp Pro                            1               5 GGT TAT ATT GTA ATT GATGTC AAC GCG GGC ACC GCG GAC AAG CCG GAC 158 Gly Tyr Ile Val Ile Asp ValAsn Ala Gly Thr Ala Asp Lys Pro Asp 10                  15                  20                  25 CCA CGTCTC CCC TCC ATG AAG CAG GGC TTC AAC CGC CGC TGG ATT GGA 206 Pro Arg LeuPro Ser Met Lys Gln Gly Phe Asn Arg Arg Trp Ile Gly                 30                  35                  40 ACT AAT ATCGAT TTC GTT TAT GTC GTG TAC ACT CCT CAA GGT GCT TGT 254 Thr Asn Ile AspPhe Val Tyr Val Val Tyr Thr Pro Gln Gly Ala Cys             45                  50                  55 ACT GCA CTT GACCGT GCT ATG GAA AAG TGT TCT CCC GGT ACA GTC AGG 302 Thr Ala Leu Asp ArgAla Met Glu Lys Cys Ser Pro Gly Thr Val Arg         60                  65                  70 ATC GTC TCT GGC GGCCAT TGC TAC GAG GAC TTC GTA TTT GAC GAA TGC 350 Ile Val Ser Gly Gly HisCys Tyr Glu Asp Phe Val Phe Asp Glu Cys     75                  80                  85 GTC AAG GCC ATC ATC AACGTC ACT GGT CTC GTT GAG AGT GGT TAT GAC 398 Val Lys Ala Ile Ile Asn ValThr Gly Leu Val Glu Ser Gly Tyr Asp 90                  95                 100                 105 GAC GATAGG GGT TAC TTC GTC AGC AGT GGA GATACA AAT TGG GGC TCC 446 Asp Asp ArgGly Tyr Phe Val Ser Ser Gly Asp Thr Asn Trp Gly Ser                110                 115                 120 TTC AAG ACCTTG TTC AGA GAC CAC GGA AGA GTT CTT CCC GGG GGT TCC 494 Phe Lys Thr LeuPhe Arg Asp His Gly Arg Val Leu Pro Gly Gly Ser            125                 130                 135 TGC TAC TCC GTCGGC CTC GGT GGC CAC ATT GTC GGC GGA GGT GAC GGC 542 Cys Tyr Ser Val GlyLeu Gly Gly His Ile Val Gly Gly Gly Asp Gly        140                 145                 150 ATT TTG GCC CGC TTGCAT GGC CTC CCC GTC GAT TGG CTC AGC GGC GTG 590 Ile Leu Ala Arg Leu HisGly Leu Pro Val Asp Trp Leu Ser Gly Val    155                 160                 165 GAG GTC GTC GTT AAG CCAGTC CTC ACC GAA GAC TCG GTA CTC AAG TAT 638 Glu Val Val Val Lys Pro ValLeu Thr Glu Asp Ser Val Leu Lys Tyr170                 175                 180                 185 GTG CACAAA GAT TCC GAA GGC AAC GAC GGG GAG CTC TTT TGG GCA CAC 686 Val His LysAsp Ser Glu Gly Asn Asp Gly Glu Leu Phe Trp Ala His                190                 195                 200 ACA GGT GGCGGT GGC GGA AAC TTT GGA ATC ATC ACC AAA TAC TAC TTC 734 Thr Gly Gly GlyGly Gly Asn Phe Gly Ile Ile Thr Lys Tyr Tyr Phe            205                 210                 215 AAG GAT TTG CCCATG TCT CCA CGG GGC GTC ATC GCA TCA AAT TTA CAC 782 Lys Asp Leu Pro MetSer Pro Arg Gly Val Ile Ala Ser Asn Leu His        220                 225                 230 TTC AGC TGG GAC GGTTTC ACG AGA GAT GCC TTG CAG GAT TTG TTG ACA 830 Phe Ser Trp Asp Gly PheThr Arg Asp Ala Leu Gln Asp Leu Leu Thr    235                 240                 245 AAG TAC TTC AAA CTT GCCAGA TGT GAT TGG AAG AAT ACG GTT GGC AAG 878 Lys Tyr Phe Lys Leu Ala ArgCys Asp Trp Lys Asn Thr Val Gly Lys250                 255                 260                 265 TTT CAAATC TTC CAT CAG GCA GCG GAA GAG TTT GTC ATG TAC TTG TAT 926 Phe Gln IlePhe His Gln Ala Ala Glu Glu Phe Val Met Tyr Leu Tyr                270                 275                 280 ACA TCC TACTCG AAC GAC GCC GAG CGC GAA GTT GCC CAA GAC CGT CAC 974 Thr Ser Tyr SerAsn Asp Ala Glu Arg Glu Val Ala Gln Asp Arg His            285                 290                 295 TAT CAT TTG GAGGCT GAC ATA GAA CAG ATC TAC AAA ACA TGC GAG CCC 1022 Tyr His Leu Glu AlaAsp Ile Glu Gln Ile Tyr Lys Thr Cys Glu Pro        300                 305                 310 ACC AAA GCG CTT GGCGGG CAT GCT GGG TGG GCG CCG TTC CCC GTG CGG 1070 Thr Lys Ala Leu Gly GlyHis Ala Gly Trp Ala Pro Phe Pro Val Arg    315                 320                 325 CCG CGC AAG AGG CAC ACATCC AAG ACG TCG TAT ATG CAT GAC GAG ACG 1118 Pro Arg Lys Arg His Thr SerLys Thr Ser Tyr Met His Asp Glu Thr330                 335                 340                 345 ATG GACTAC CCC TTC TAC GCG CTC ACT GAG ACG ATC AAC GGC TCC GGG 1166 Met Asp TyrPro Phe Tyr Ala Leu Thr Glu Thr Ile Asn Gly Ser Gly                350                 355                 360 CCG AAT CAGCGC GGC AAG TAC AAG TCT GCG TAC ATG ATC AAG GAT TTC 1214 Pro Asn Gln ArgGly Lys Tyr Lys Ser Ala Tyr Met Ile Lys Asp Phe            365                 370                 375 CCG GAT TTC CAGATC GAC GTG ATC TGG AAA TAC CTT ACG GAG GTC CCG 1262 Pro Asp Phe Gln IleAsp Val Ile Trp Lys Tyr Leu Thr Glu Val Pro        380                 385                 390 GAC GGC TTG ACT AGTGCC GAA ATG AAG GAT GCC TTA CTC CAG GTG GAC 1310 Asp Gly Leu Thr Ser AlaGlu Met Lys Asp Ala Leu Leu Gln Val Asp    395                 400                 405 ATG TTT GGT GGT GAG ATTCAC AAG GTG GTC TGG GAT GCG ACG GCA GTC 1358 Met Phe Gly Gly Glu Ile HisLys Val Val Trp Asp Ala Thr Ala Val410                 415                 420                 425 GCG CAGCGC GAG TAC ATC ATC AAA CTG CAG TAC CAG ACA TAC TGG CAG 1406 Ala Gln ArgGlu Tyr Ile Ile Lys Leu Gln Tyr Gln Thr Tyr Trp Gln                430                 435                 440 GAA GAA GACAAG GAT GCA GTG AAC CTC AAG TGG ATT AGA GAC TTT TAC 1454 Glu Glu Asp LysAsp Ala Val Asn Leu Lys Trp Ile Arg Asp Phe Tyr            445                 450                 455 GAG GAG ATG TATGAG CCG TAT GGC GGG GTT CCA GAC CCC AAC ACG CAG 1502 Glu Glu Met Tyr GluPro Tyr Gly Gly Val Pro Asp Pro Asn Thr Gln        460                 465                 470 GTG GAG AGT GGT AAAGGT GTG TTT GAG GGA TGC TAC TTC AAC TAC CCG 1550 Val Glu Ser Gly Lys GlyVal Phe Glu Gly Cys Tyr Phe Asn Tyr Pro    475                 480                 485 GAT GTG GAC TTG AAC AACTGG AAG AAC GGC AAG TAT GGT GCC CTC GAA 1598 Asp Val Asp Leu Asn Asn TrpLys Asn Gly Lys Tyr Gly Ala Leu Glu490                 495                 500                 505 CTT TACTTT TTG GGT AAC CTG AAC CGC CTC ATC AAG GCC AAA TGG TTG 1646 Leu Tyr PheLeu Gly Asn Leu Asn Arg Leu Ile Lys Ala Lys Trp Leu                510                 515                 520 TGG GAT CCCAAC GAG ATC TTC ACA AAC AAA CAG AGC ATC CCT ACT AAA 1694 Trp Asp Pro AsnGlu Ile Phe Thr Asn Lys Gln Ser Ile Pro Thr Lys            525                 530                 535 CCT CTT AAG GAGCCC AAG CAG ACG AAA TAGTAGGTCA CAATTAGTCA 1741 Pro Leu Lys Glu Pro LysGln Thr Lys         540                 545 TCGACTGAAG TGCAGCACTTGTCGGATACG GCGTGATGGT TGCTTTTTAT AAACTTGGTA 1801

In the amino acid sequence shown in the above Table 3.2., the HOX-1 toHOX-8 peptides are shown with bolded or underlined codes. Bolded codesindicate amino residues which have been confirmed by amino acidsequencing of the peptides. The underlined codes indicate amino acidresidues which are derived from the nucleotide sequence, but which havenot been confirmed by sequencing of the relevant HOX peptides.

HOX-1 is amino acid residues 461-468, HOX-2 residues 92-114, HOX-3residues 219-234, HOX-4 residues 189-202, HOX-5 residues 215-218, HOX-6residues 8-22, HOX-7 residues 434-444 and HOX-8 residues 452-460.

EXAMPLE 4

Production of Recombinant Hexose Oxidase in Pichia pastoris

4.1. Construction of a Vector For The Expression of Recombinant HexoseOxidase in Pichia pastoris

The open reading frame encoding Chondrus crispus hexose oxidase as shownin Table 3.2. was inserted into a Pichia pastoris expression vector,pPIC3 (Research Corporation Technologies, Inc., Tucson, Ariz.85711-3335). The plasmid contains the alcohol oxidase promotor (aox1promotor) and transcriptional termination signal from Pichia pastoris(in FIG. 9, aoxp and aoxt, respectively). A his4⁺ gene in the vectorenables selection of His⁺ recombinant Pichia pastoris cells. When thisexpression cassette is transformed into Pichia pastoris it integratesinto the chromosomal DNA. Pichia pastoris cells harbouring an expressioncassette with a Chondrus crispus hexose oxidase gene inserted downstreamof the aox1 promotor can be induced to produce hexose oxidase by theaddition of the inducer of the aox1 promotor, methanol. A mutant ofPichia pastoris, KM71, which is defective in the major alcohol oxidasegene, aox1, can be used as recipient of the hexose oxidase gene (Creggand Madden 1987; Tschopp et al. 1987). However, Pichia pastoris containsanother alcohol oxidase gene, aox2, which can also be induced bymethanol. Thus, recombinant Pichia pastoris transformed with a hexoseexpression cassette will produce two oxidases, hexose oxidase andalcohol oxidase, upon addition of methanol.

Before insertion of the hexose oxidase gene into the expression vectorpPIC3, sequences 5′ and 3′ of the open reading were modified. Firststrand cDNA was used as template in PCR. The synthetic oligonucleotidespecific for the 5′-end of the open reading frame, Hox5′-1 (Table 3.1)was used as PCR-primer together with a primer (Hox3′-1) specific for the3′-end of the sequence encoding Chondrus crispus hexose oxidase. Theprimer Hox3′-1 had the sequence 5′-ACCAAGTTTATAAAAAGCAACCATCAC-3′ (SEQID NO:32). PCR amplification was carried out using the GeneAmp® PCRReagent Kit with AmpliTaq® DNA polymerase (Perkin-Elmer Cetus). The PCRprogram was 30 cycles at 30 sec at 94° C., 30 sec at 55° C. and 2 min at72° C. Gel electrophoresis of the reaction mixture showed a band withthe approximate size of 1.7 kb. This 1.7 kb fragment was inserted intothe vector pT7 Blue (Novagen) (plasmid pUPO150) and subjected to DNAsequencing.

The fragment encoding Chondrus crispus hexose oxidase was furthersubcloned into the Pichia pastoris expression vector pPIC3 (Clare et al.1991) as shown in FIG. 9. Plasmid pT7 Blue harbouring the hexose oxidasegene was restricted with the restriction endonuclease NdeI and the endswere polished with Klenow DNA polymerase essentially as described bySambrook et al. (1989). After heat inactivation of the DNA polymerase(Sambrook et al. 1989) the DNA was restricted further with EcoRI and theDNA fragment containing the hexose oxidase gene was purified on anagarose gel as a blunt end—EcoRI DNA fragment (QIAEX™, QIAGEN).

The Pichia pastoris expression vector pPIC3 was restricted with therestriction enzymes SnaBI and EcoRI and purified on an agarose gel. Thepurified vector and the fragment encoding hexose oxidase were ligatedand the ligation mixture was transformed into E. coli DH5α (LifeTechnologies), essentially as described by Sambrook et al. (1989). Theresulting expression vector containing the hexose oxidase gene fromChondrus crispus, plasmid pUPO153, was subjected to DNA sequencing toensure that no mutations had occurred in the hexose oxidase gene duringthe subcloning procedure.

Plasmid pUPO153 was purified from E. coli DH5α and introduced intoPichia pastoris using electroporation (The Pichia Yeast ExpressionSystem, Phillips Petroleum Company) or using The Pichia SpheroplastModule (Invitrogen, San Diego, USA, cat. no. K1720-01). Themethanol-utilization-defective mutant of Pichia pastoris, KM71 (genotypehis4, aox1::ARG4), (Cregg and Madden 1987; Tschopp et al. 1987) was usedas recipient. Recombinant Pichia pastoris colonies selected on agarplates without histidine were screened for the presence of the hexoseoxidase gene using PCR. Primers specific for hexose oxidase (Table 3.1)were used in addition to primers specific for the Pichia pastorisalcohol oxidase promoter and transcription termination signal(Invitrogen, cat. nos. N710-02 and N720-02, respectively).

A sample of Pichia pastoris KM71 containing pUPO153 was deposited withthe Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM),Mascheroder Weg 1b, D-38124 Braunschweig, Germany on 23 May 1996 underthe accession number DSM 10693.

4.2. Expression of Recombinant Hexose Oxidase in Pichia Pastoris

Pichia pastoris strain KM71 containing the expression cassette with thehexose oxidase gene inserted between the aox1 promoter and thetranscription termination signal was cultivated in shake flasks in MD(1.34 grams per liter of yeast nitrogen base (Difco, cat. no.0919-15-3), 0.4 mg/l of biotin, 0.1% arginine, and 20 g/l glucose).One-liter shake flasks containing 150 ml culture were incubated in arotary shaker at 30° C., 300 rpm. When the cells reached a density ofOD₆₀₀=15−20 the cells were harvested by centrifugation at 6,000×g for 10min and resuspended in a similar volume (150 ml) of induction medium, MM(1.34 g/l of yeast nitrogen base, 0.4 mg/l of biotin, 0.1% arginine, and1% methanol). After growth for two days, additional methanol (0.5%) wasadded to compensate for the consumption and evaporation of methanol.

Three or four days after induction the cells were harvested bycentrifugation (6,000×g, 10 min) and resuspended in about ⅕ of thegrowth volume of 50 mM Tris-Cl, pH 7.5. Resuspended cells were kept colduntil disrupture in a FRENCH® Press (SLM Instruments, Inc., Rochester,N.Y.).

Cells were opened in a 20K FRENCH® Pressure Cell at an internal pressureof 20,000 psi. The cell extract was cleared by centrifugation at10,000×g for 10 min at 5° C. The hexose oxidase containing supernatantwas carefully removed and subjected to purification as described below.

4.3. Purification of Recombinant Hexose Oxidase from Pichia Pastoris

4.3.1. First Step, Anion Exchange Chromatography.

Clarified homogenate from FRENCH press homogenization (100-150 ml) wassubjected to anion exchange chromatography on an FPLC system equippedwith two 5-ml HiTrap-Q columns prepacked with Q-Sepharose HighPerformance (Pharmacia). The columns were connected in series and thechromatography was carried out at room temperature. The column wasequilibrated in buffer A: 20 mM Tris-Cl, pH 7,5. The flow rate was 1.25ml during sample application and 2.5 ml during wash and elution. Aftersample application the column was washed with 30 ml of buffer A.Adsorbed proteins were then eluted with 200 ml of a gradient from bufferA to buffer B: 20 mM Tris-Cl, 750 mM NaCl, pH 7.5. Fractions of 2 mlwere collected during wash and gradient elution. The fractions wereassayed for hexose oxidase activity as described above in Example 1.3(10 μl of sample, 15 min of incubation time). The fractions were alsoassayed for alcohol oxidase (AOX) activity in an assay which wasidentical to the hexose oxidase assay except that 0.5% methanol insteadof 0.05 M glucose was used as substrate. As seen in FIG. 10, theactivity profiles showed that AOX and HOX co-eluted at a saltconcentration of about 400 mM NaCl. Fractions containing hexose oxidasewere pooled and stored at 4° C.

4.3.2. Second Step, Gel Filtration.

The pool from step one in the purification (20-30 ml) was concentratedto about 3.5 ml by centrifugal ultracentrifugation at 4° C. inCentriprep concentrators (Amicon, USA, nominal molecular weight cut-off30,000). The concentrated preparation of hexose oxidase was clarified bycentrifugation and the supernatant was mixed with glycerol to a finalconcentration of 5%. The sample was applied onto the column using anSA-5 sample applicator (Pharmacia) connected to the inlet of the column.Gel filtration was carried out at 4° C. on an XK 26/70 column (2.6×66cm, Pharmacia) with a bed volume of 350 ml. The column was packed withSephacryl S-200 HR (Pharmacia) according to the instructions of themanufacturer. The buffer was 20 mM Tris-Cl, 500 mM NaCl, pH 7.5 and theperi-staltic P1-pump (Pharmacia) was set at 0.5 ml/min. TheUV-absorbance at 280 nm was recorded. Fractions of 2.5 ml were collectedand assayed for hexose oxidase and alcohol oxidase activity as describedabove (10 μl of sample, 15 min of incubation time). The activityprofiles clearly showed that AOX and HOX activities were separated, seeFIG. 11. This result of the gel filtration was expected since alcoholoxidase from methylotrophic yeasts like Pichia pastoris have a nativemolecular weight of about 600,000 (Sahm & Wagner, 1973), whereas HOX hasa native molecular weight of about 110,000-130,000, as described insection 1.8. The elution volume of recombinant HOX was identical to theelution volume observed earlier on the same column for native HOX fromChondrus crispus (section 1.7 and 1.8). Thus, recombinant HOX appearedto be of the same molecular weight as native HOX isolated directly fromChondrus crispus. Fractions containing hexose oxidase were pooled andstored at 4° C.

4.4.3. Third Step, Anion Exchange Chromatography on Mono Q Column.

The pool from the above second step was further purified by anionexchange chromatography on a FPLC system equipped with a Mono Q HR 5/5column (bed volume 1 ml). The column was equilibrated in buffer A: 20 mMTris-Cl, pH 7,5. The flow rate was 1 ml/min. The pool from step two wasdesalted by gel filtration in buffer A on pre-packed Sephadex G-25columns (PD-10, Pharmacia). After sample application the column waswashed with 30 ml of buffer A. Adsorbed proteins were eluted with 20 mlof a gradient from 0% to 100% buffer B: 20 mM Tris-Cl, 500 mM NaCl, pH7,5. Fractions of 0,5 ml were collected and assayed for hexose oxidaseactivity as described above (10 μl of sample, 15 min of incubationtime). Fractions containing hexose oxidase were pooled and stored at 4°C.

4.3.4. Fourth Step, Chromatofocusing.

The pool from the above third step was purified by chromatofocusing on aMono P HR 5/5 column as described above in Example 1.13, except that thePhenyl Sepharose adsorption step was omitted. When comparing native andrecombinant hexose oxidase—both forms obtained by a final purificationby chromatofocusing—it was found that the specific activity ofrecombinant hexose oxidase from Pichia pastoris was similar to that ofthe native form isolated from Chondrus crispus. Fractions containinghexose oxidase were analyzed by SDS-PAGE and staining of the gel withCoomassie Brilliant Blue R-250 as described above. The purifiedpreparation of recombinant hexose oxidase was composed of two bandsmigrating at 40 kD and 29 kD, respectively.

In conclusion, recombinant hexose oxidase could be isolated and purifiedfrom the host organism Pichia pastoris. In SDS-PAGE the recombinant,purified enzyme exhibited the same bands at 40 kD and 29 kD as thecorresponding native enzyme from Chondrus crispus.

4.4. Properties of Recombinant Hexose Oxidase from Pichia pastoris

Generation and amino acid sequence analysis of peptide fragments ofrecombinant hexose oxidase (rHOX).

Purified rHOX was used for preparative SDS-PAGE and electro-blotting toPVDF membrane, as described above in Example 2.4.

The resulting 40 kD and 29 kD bands were subjected to enzymaticdigestion of PVDF-bound hexose oxidase polypeptides, as described abovein Example 2.5. The peptide fragments were separated by reversed-phaseliquid chromatography as described above in Example 2.7. Well-resolvedand abundant peptides were selected for amino acid sequence analysis byautomated Edman degradation (10 steps), as described above in Example2.3. The obtained amino acid sequences are shown in Table 4.1.

TABLE 4.1 Peptide sequences obtained by sequence analysis ofendoproteinase Lys-C peptides derived from 40 kD and 29 kD polypeptidesof recombinant hexose oxidase expressed in Pichia pastoris Origin ofSequence sequenced identification Amino acid sequence peptide Step no. 12 3 4 5 6 7 8 9 10 40 kD HOX-9 D P G Y I V I D V N (SEQ ID NO: 35)peptide 29 kD HOX-10 L Q Y Q T Y W Q E E (SEQ ID NO: 36) peptide and Yand L and T and E and V and P and D and G and L and T (SEQ ID NO: 37)

The HOX-9 peptide sequence from the recombinant 40 kD polypeptide showeda sequence identical to Aspx₈ through Asn₁₇ in the amino acid sequenceof hexose oxidase from Chondrus crispus as shown in Table 3.2. (SEQ IDNO:30). Sequence analysis of a peptide sample obtained from therecombinant 29 kD polypeptide showed two residues at each step. Theamino acid identifications showed that two peptides present in thesample correspond to Leu₄₃₄ through Glu₄₄₃ and Tyr₃₈₈ through Thr₃₉₇,respectively, in the amino acid sequence of hexose oxidase from Chondruscrispus, see Table 3.2. (SEQ ID NO:30).

It could thus be concluded that the peptide sequences obtained fromrecombinant hexose oxidase were identical to the corresponding aminoacid sequence of native hexose oxidase from Chondrus crispus.

Furthermore, it could be concluded that Pichia pastoris transformed withthe hexose oxidase gene from Chondrus crispus was capable of producingrecombinant hexose oxidase.

4.4.1. Substrate Specificity

The substrate specificity of recombinant hexose oxidase from Pichiapastoris and native hexose oxidase from Chondrus crispus was comparedusing a number of sugars at a final concentration of 0.1 M in the assaydescribed above. The relative rates are shown in Table 4.2.

TABLE 4.2 Substrate specificity of recombinant hexose oxidase expressedin Pichia pastoris and native hexose oxidase from Chondrus crispusRelative rate native enzyme, recombinant native enzyme, Sullivan andIkawa, Substrate enzyme this work 1973 D-Glucose 100 100 100 D-Galactose75 75 82 Maltose 57 37 40 Cellobiose 51 33 32 Lactose 38 25 22

As shown in Table 4.2., the substrate specificity of recombinant hexoseoxidase was almost identical to that of the native enzyme. However,although the relative rate among disaccharides decreased for both enzymeforms in the order maltose, cellobiose and lactose, the recombinantenzyme appeared to be less selective in its ability to oxidize thesedisaccharides. The results for the native enzyme were almost identicalto the data reported earlier by Sullivan et al. (1973).

4.4.2. Inhibition by Sodium Diethyldithiocarbamate

Sullivan and Ikawa (1973) reported that hexose oxidase from Chondruscrispus is strongly inhibited by sodium diethyldithiocarbamate.Recombinant hexose oxidase from Pichia pastoris was compared to thenative enzyme from Chondrus crispus with respect to inhibition by thiscopper-binding compound. The inhibitor was included in the enzyme assayin two concentrations, 0.1 mM and 0.01 mM, as described by Sullivan andIkawa (1973). The results are summarized in Table 4.3.

TABLE 4.3 Comparison of the inhibitory effect of sodiumdiethyldithiocarbamate on the enzymatic activity of recombinant hexoseoxidase from Pichia pastoris and native hexose oxidase from Chondruscrispus Inhibition (%) Concentration of Recombinant Native inhibitorenzyme enzyme  0.1 mM 96 95 0.01 mM 39 41

It appears from Table 4.3 that recombinant and native hexose oxidasewere equally sensitive when subjected to inhibition by sodiumdiethyldithiocarbamate. Furthermore, the results were similar to thedata for native hexose oxidase reported by Sullivan and Ikawa (1973).

EXAMPLE 5

Production of Recombinant Hexose Oxidase in Escherichia coli

5.1. Construction of a Vector for the Expression of Recombinant HexoseOxidase in Escherichia coli

The open reading frame encoding Chondrus crispus hexose oxidase shown inTable 3.2. (SEQ ID NO:30) was inserted into an Escherichia coliexpression vector, pET17b (Novagen, cat. no. 69726-1). The plasmidcontains a strong inducible bacteriophage T7 promotor and a T7transcription termination signal. Genes inserted between thesecontrolling elements can be expressed by the addition of isopropylβ-D-thiogalactopy-ranoside (IPTG) if the plasmid is propagated inspecial E. coli host cells e.g. strain BL21(DE3) (Novagen, cat. no.69387-1).

The hexose oxidase gene was modified at the 5′ and 3′ ends in order toinsert the gene in the expression vector pET17b. The hexose oxidase genewas isolated by PCR with primers specific for the 5′ and 3′ ends of thehexose oxidase gene. The 5′ primer (Hox5′-2) had the DNA sequence5′-ATGAATTCGTGGGTCGAAGAGCCC-3′ (SEQ ID NO:33) and the primer specificfor the 3′-end was Hox3′-1. First strand cDNA from Chondrus crispus wasused as template. PCR amplification was carried out with AmpliTaq® DNApolymerase (Perkin-Elmer Cetus) as described in example 4.1. Gelelectrophoresis of the reaction mixture showed a band with theapproximate size of 1.7 kb. This 1.7 kb fragment was inserted into thevector pT7 Blue (Novagen) giving rise to plasmid pUPO161.

Modification of the 5′-end of the hexose oxidase gene and furthersubcloning of the gene into the E. coli expression vector is shown inFIG. 12. The 5′-end was modified by PCR in order to insert a NdeI siteright at the ATG translation start. The oligonucleotide, Hox5′-4, withthe sequence 5′-CAGGAATTCATATGGCTACTCTTCCCCAGAAAG-3′ (SEQ ID NO:34) wasused together with the oligonucleotide Hox13− (SEQ ID NO:28) (Table3.1). PCR amplification was as described above in Example 4.1. Thereaction mixture was fractionated on a 2% agarose gel and the hexoseoxidase specific 180 bp fragment was purified as described in Example3.4. The 180 bp fragment was restricted with the restrictionendonuclease ClaI and EcoRI and ligated to pUPO161 restricted with thesame enzymes giving rise to plasmid pUPO167.

The hexose oxidase gene in plasmid pUPO167 was further subcloned inorder to construct a hexose oxidase expression vector for E. coli.Plasmid pUPO167 was restricted with the enzymes NdeI and BamHI and withthe enzymes BamHI and SalI. The first reaction gave rise to a 1.6 kbfragment encoding the 5′ and the middle part of the hexose oxidase genewhile the reaction with the enzymes BamHI and SailI gave a 200 bpfragment encoding the 3′ end of the hexose oxidase gene. The two hexoseoxidase specific fragments were purified on agarose gels as described inExample 3.4 and ligated to plasmid pET17b restricted with therestriction endonucleases NdeI and XhoI. Plasmid pET17b harbouring thehexose oxidase gene was denoted pUPO181. DNA sequencing showed that nomutation was introduced in the hexose oxidase gene during the isolationand cloning process.

5.2. Expression of Recombinant Hexose Oxidase in Escherichia coli

Plasmid pUPO181 was introduced into E. coli strain BL21(DE3) (Novagen)by a standard transformation procedure (Sambrook et al. 1989). The cellswere grown in shake flasks in LB medium (Sambrook et al. supra). At acell density of OD₆₀₀=0.5 the cells were induced to express recombinanthexose oxidase by the addition of 10⁻³ M IPTG. One hour after theaddition of IPTG the cells were harvested by centrifugation andresuspended in sample buffer and subjected to SDS-PAGE as describedabove in Example 1.10.

The result of the electrophoresis is shown in FIG. 13. The crude extractof E. coli expressing recombinant hexose oxidase enzyme from plasmidpUPO181 showed a prominent protein band at Mr 62 kD. This 62 kD band hadthe same molecular weight as the translation product predicted from theopen reading frame. Non-transformed E. coli cells showed no such 62 kDprotein.

A sample of E. coli BL21(DE3) containing pUPO181 was deposited with theDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM),Mascheroder Weg 1b, D-38124 Braunschweig, Germany on 23 May 1996 underthe accession number DSM 10692.

EXAMPLE 6

Production of Recombinant Hexose Oxidase in Saccharomyces cerevisiae

6.1. Construction of a Vector for the Expression of Recombinant HexoseOxidase in Saccharomyces cerevisiae

The open reading frame encoding Chondrus crispus hexose oxidase shown inTable 3.2. (SEQ ID NO:30) was inserted into a yeast expression vector,pYES2 (Invitrogen, cat. no. V825-20). Plasmid pYES2 is a high-copynumber episomal vector designed for inducible expression of recombinantproteins in Saccharomyces cerevisiae. The vector contains upstreamactivating and promoter sequences from the S. cerevisiae Gal1 gene forhigh-level, tightly regulated transcription. The transcriptiontermination signal is from the CYC1 gene.

The hexose oxidase gene from Chondrus crispus was modified at the 5′-and 3′-ends in order to insert the gene in the expression vector pYES2.The hexose oxidase gene was isolated from plasmid pUPO150 as describedin Example 4.1 (FIG. 9). The hexose oxidase gene was isolated on a bluntend-EcoRI DNA fragment as described and inserted into plasmid pYES2restricted with the enzymes PvuII and EcoRI (FIG. 14). The resultingplasmid, pUPO155, was subjected to DNA sequencing in order to show thatno mutation had occurred during the cloning procedure.

Plasmid pUPO155 was purified from E. coli DH5α and transformed into S.cerevisiae by electroporation (Grey and Brendel 1992). The strainPAP1500 (genotype α, ura3-52, trp1::GAL10-GAL4, lys2-801, leu2Δ1,his3Δ200, pep4::HIS3, prb1Δ1.6R, can1, GAL) (Pedersen et al. 1996) wasused as a recipient.

6.2. Expression of Recombinant Hexose Oxidase in Saccharomycescerevisiae

S. cerevisiae strain 1500 containing plasmid pUPO155 was grown andinduced with 2% galactose as described by Pedersen et al. (1996). Threedays after the induction the cells were harvested by centrifugation andlysed as described above in Example 4.2. The crude extract was assayedfor hexose oxidase activity using the o-dianisidine assay describedabove in Example 1.3. Table 6.1 shows that S. cerevisiae cellsharbouring the hexose oxidase gene are capable of expressing activehexose oxidase.

TABLE 6.1 Production of recombinant hexose oxidase in Saccharomycescerevisiae Saccharomyces cerevisiae +hexose non-recombinant Substrateoxidase gene control D-Glucose ++ 0 D-Galactose + 0 no substrate 0 0 0 =no detectable activity

A sample of S. cerevisiae strain 1500 containing plasmid pUPO155 wasdeposited with the Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSM), Mascheroder Weg 1b, D-38124 Braunschweig,Germany on 23 May 1996 under the accession number DSM 10694.

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1. An isolated DNA fragment encoding a polypeptide having hexose oxidaseactivity, wherein said polypeptide oxidizes at least maltose and whereinsaid DNA fragment consists of the nucleic acid sequence from position 84through position 1721 of SEQ ID NO:30.
 2. The DNA fragment according toclaim 1 wherein the DNA fragment is operably linked to an expressionsignal not natively associated with said DNA fragment.
 3. An isolatedhost cell comprising the DNA fragment of claim
 2. 4. The host cellaccording to claim 3 which is a microbial cell selected from the groupconsisting of a bacterial cell, a fungal cell and a yeast cell.
 5. Thehost cell according to claim 4 selected from the group consisting of anEscherichia coli cell, a lactic acid bacterial cell, a Saccharomycescerevisiae cell and a Pichia pastoris cell.
 6. A method for producing apolypeptide having hexose oxidase activity, said method comprisingcultivating in a cultivating medium the host cell of claim 3 said hostcell comprising the DNA fragment operably linked to an expressionsignal, and recovering the enzyme from the cells and/or the culturingmedium.
 7. The method according to claim 6 wherein the host cell is amicroorganism selected from the group consisting of a bacterial species,a fungal species and a yeast species.
 8. The method according to claim 7wherein the host cell is selected from the group consisting of an E.coli cell, a lactic acid bacterial cell, a S. cerevisiae cell and a P.pastoris cell.
 9. The method according to claim 6 comprising as afurther step a purification of the polypeptide preparation initiallyrecovered from the cultivation medium and/or the host cells to obtain apreparation in which the polypeptide is in a substantially pure form.10. The method according to claim 6 wherein the polypeptide havinghexose oxidase activity is a fusion product.
 11. A dough improvingcomposition comprising the host cell of claim 3 capable of expressingsuch a polypeptide in dough and at least one conventional doughcomponent.
 12. The composition according to claim 11, further comprisingat least one enzyme selected from the group consisting of a cellulase, ahemicellulase, a xylanase, a pentosanase, an amylase, a lipase and aprotease.